FcGammaRIIB Specific Antibodies and Methods of Use Thereof

ABSTRACT

The present invention relates to humanized FcγRIIB antibodies, fragments, and variants thereof that bind human FcγRIIB with a greater affinity than said antibody binds FcγRIIA. The invention encompasses the use of the humanized antibodies of the invention for the treatment of any disease related to loss of balance of Fc receptor mediated signaling, such as cancer (preferably a B-cell malignancy, particularly, B-cell chronic lymphocytic leukemia or non-Hodgkin&#39;s lymphoma), autoimmune disease, inflammatory disease or IgE-mediated allergic disorder. The present invention also encompasses the use of a humanized FcγRIIB antibody or an antigen-binding fragment thereof, in combination with other cancer therapies. The invention provides methods of enhancing the therapeutic effect of therapeutic antibodies by administering the humanized antibodies of the invention to enhance the effector function of the therapeutic antibodies. The invention also provides methods of enhancing the efficacy of a vaccine composition by administering the humanized antibodies of the invention with a vaccine composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/126,978 filed on May 10, 2005, which claims priority to U.S.Provisional Application No. 60/582,043, filed on Jun. 21, 2004, and U.S.Provisional Application No. 60/569,882, filed May 10, 2004. All of theabove-identified applications are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies or fragments thereof thatspecifically bind FcγRIIB, particularly human FcγRIIB, with greateraffinity than said antibodies or fragments thereof bind FcγRIIA,particularly human FcγRIIA. The present invention also encompasses theuse of an anti-FcγRIIB antibody or an antigen-binding fragment thereof,as a single agent therapy for the treatment, prevention, management, oramelioration of a cancer, preferably a B-cell malignancy, particularly,B-cell chronic lymphocytic leukemia or non-Hodgkin's lymphoma, anautoimmune disorder, an inflammatory disorder, an IgE-mediated allergicdisorder, or one or more symptoms thereof. The present invention alsoencompasses the use of an anti-FcγRIIB antibody or an antigen-bindingfragment thereof, in combination with other cancer therapies. Thepresent invention provides pharmaceutical compositions comprising ananti-FcγRIIB antibody or an antigen-binding fragment thereof, in amountseffective to prevent, treat, manage, or ameliorate a cancer, such as aB-cell malignancy, an autoimmune disorder, an inflammatory disorder, anIgE-mediated allergic disorder, or one or more symptoms thereof. Theinvention further provides methods of enhancing the therapeutic effectof therapeutic antibodies by administering the antibodies of theinvention to enhance the effector function of the therapeuticantibodies. The invention also provides methods of enhancing efficacy ofa vaccine composition by administering the antibodies of the inventionwith a vaccine composition.

BACKGROUND OF THE INVENTION I. Fc Receptors and their Roles in theImmune System

The interaction of antibody-antigen complexes with cells of the immunesystem results in a wide array of responses, ranging from effectorfunctions such as antibody-dependent cytotoxicity, mast celldegranulation, and phagocytosis to immunomodulatory signals such asregulating lymphocyte proliferation and antibody secretion. All theseinteractions are initiated through the binding of the Fc domain ofantibodies or immune complexes to specialized cell surface receptors onhematopoietic cells. The diversity of cellular responses triggered byantibodies and immune complexes results from the structuralheterogeneity of Fc receptors. Fc receptors share structurally relatedligand binding domains which presumably mediate intracellular signaling.

The Fc receptors, members of the immunoglobulin gene superfamily ofproteins, are surface glycoproteins that can bind the Fc portion ofimmunoglobulin molecules. Each member of the family recognizesimmunoglobulins of one or more isotypes through a recognition domain onthe a chain of the Fc receptor. Fc receptors are defined by theirspecificity for immunoglobulin subtypes. Fc receptors for IgG arereferred to as FcγR, for IgE as FcεR, and for IgA as FcαR. Differentaccessory cells bear Fc receptors for antibodies of different isotype,and the isotype of the antibody determines which accessory cells will beengaged in a given response (reviewed by Ravetch J. V. et al. (1991) “FcReceptors,” Annu. Rev. Immunol. 9: 457-92; Gerber et al. (2001)“Stimulatory And Inhibitory Signals Originating From The MacrophageFcgamma Receptors,” Microbes and Infection, 3: 131-139; Billadeau et al.(2002), “ITAMs Versus ITIMs: Striking A Balance During Cell Regulation,”The Journal of Clinical Investigation, 2(109): 161-168; Ravetch J. V. etal. (2000) “Immune Inhibitory Receptors,” Science, 290: 84-89; Ravetchet al. (2001) “IgG Fc Receptors,” Annu. Rev. Immunol. 19:275-290;Ravetch (1994) “Fc Receptors: Rubor Redux,” Cell, 78(4): 553-560). Thedifferent Fc receptors, the cells that express them, and their isotypespecificity is summarized in Table 1 (adapted from IMMUNOBIOLOGY: THEIMMUNE SYSTEM IN HEALTH AND DISEASE, 4^(th) ed. 1999, Elsevier ScienceLtd/Garland Publishing, New York).

A. Fcγ Receptors

Each member of this family is an integral membrane glycoprotein,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, a single membrane spanning domain and anintracytoplasmic domain of variable length. There are three known FcγRs,designated FcγRI(CD64), FcγRII(CD32), and FcγRIII(CD16). The threereceptors are encoded by distinct genes; however, the extensive homologyamong the three family members suggests they arose from a commonprogenitor, perhaps by gene duplication. This invention specificallyfocuses on FcγRII(CD32).

B. FcγRII(CD32)

FcγRII proteins are 40KDa integral membrane glycoproteins that bind onlythe complexed IgG due to a low affinity for monomeric Ig (10⁶ M⁻¹). Thisreceptor is the most widely expressed FcγR, present on all hematopoieticcells, including monocytes, macrophages, B cells, NK cells, neutrophils,mast cells, and platelets. FcγRII has only two immunoglobulin-likeregions in its immunoglobulin binding chain and hence a much loweraffinity for IgG than FcγRI. There are three human FcγRII genes(FcγRII-A, FcγRII-B, FcγRII-C), all of which bind IgG in aggregates orimmune complexes.

Distinct differences within the cytoplasmic domains of FcγRII-A (CD32A)and FcγRII-B (CD32B) create two functionally heterogenous responses toreceptor ligation. The fundamental difference is that the A isoforminitiates intracellular signaling leading to cell activation such asphagocytosis and respiratory burst, whereas the β isoform initiatesinhibitory signals, e.g., inhibiting B-cell activation.

C. Signaling Through FcγRs

Both activating and inhibitory signals are transduced through the FcγRsfollowing ligation. These diametrically opposing functions result fromstructural differences among the different receptor isoforms. Twodistinct domains within the cytoplasmic signaling domains of thereceptor called Immunoreceptor Tyrosine based Activation Motifs (ITAMs)or Immunoreceptor Tyrosine based Inhibitory Motifs (ITIMS) account forthe different responses. The recruitment of different cytoplasmicenzymes to these structures dictates the outcome of the FcγR-mediatedcellular responses. ITAM-containing FcγR complexes include FcγRI,FcγRIIA, FcγRIIIA, whereas ITIM-containing complexes only includeFcγRIIB.

Human neutrophils express the FcγRIIA gene. FcγRIIA clustering viaimmune complexes or specific antibody cross-linking serves to aggregateITAMs along with receptor-associated kinases that facilitate ITAMphosphorylation. ITAM phosphorylation serves as a docking site for Sykkinase, activation of which results in activation of downstreamsubstrates (e.g., PI₃K). Cellular activation leads to release ofpro-inflammatory mediators.

The FcγRIIB gene is expressed on B lymphocytes; its extracellular domainis 96% identical to FcγRIIA and binds IgG complexes in anindistinguishable manner. The presence of an ITIM in the cytoplasmicdomain of FcγRIIB defines this inhibitory subclass of FcγR. Recently themolecular basis of this inhibition was established. When colligatedalong with an activating FcγR, the ITIM in FcγRIIB becomesphosphorylated and attracts the SH2 domain of the inositol polyphosphate5′-phosphatase (SHIP), which hydrolyzes phosphoinositol messengers thatare released as a consequence of ITAM-containing FcγR-mediated tyrosinekinase activation, thus preventing the influx of intracellular Ca⁺⁺. Inthis manner, crosslinking of FcγRIIB dampens the activating response toFcγR ligation and inhibits cellular responsiveness. B cell activation, Bcell proliferation and antibody secretion is aborted.

TABLE 1 Receptors for the Fc Regions of Immunoglobulin Isotypes Effectof Receptor Binding Cell Type Ligation FcγRI IgG1 Macrophages,Neutrophils, Uptake (CD64 10⁸ M⁻¹ Eosinophils, Dendritic cellsStimulation Activation of respiratory burst Induction of killingFcγRII-A IgG1 Macrophages, Neutrophils, Uptake (CD32) 2 × 10⁶ M⁻¹Eosinophils, Dendritic cells, Granule Platelets, Langerhan cells releaseFcγRII-B2 IgG1 Macrophages, Neutrophils, Uptake (CD32) 2 × 10⁶ M⁻¹Eosinophils Inhibition of Stimulation FcγRII-BI IgG1 B cells, Mast cellsNo uptake (CD32) 2 × 10⁶ M⁻¹ Inhibition of Stimulation FcγRIII IgG1 NKcells, Eosinophil Induction of (CD16) 5 × 10⁵ M⁻¹ macrophages,Neutrophils, Killing Mast Cells FcεRI IgG1 Mast cells, EosinophilSecretion of 10¹⁰ M⁻¹ Basophils granules FcαRI IgG1, IgA2 Macrophages,Neutropils Uptake (CD89) 10⁷ M⁻¹ Eosinophils Induction of killing

II. Diseases of Relevance

A. Cancer

A neoplasm, or tumor, is a neoplastic mass resulting from abnormaluncontrolled cell growth which can be benign or malignant. Benign tumorsgenerally remain localized. Malignant tumors are collectively termedcancers. The term “malignant” generally means that the tumor can invadeand destroy neighboring body structures and spread to distant sites tocause death (for review, see Robbins and Angell, 1976, BASIC PATHOLOGY,2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-122). Cancer can arisein many sites of the body and behave differently depending upon itsorigin. Cancerous cells destroy the part of the body in which theyoriginate and then spread to other part(s) of the body where they startnew growth and cause more destruction.

More than 1.2 million Americans develop cancer each year. Cancer is thesecond leading case of death in the United States and if current trendscontinue, cancer is expected to be the leading cause of the death by theyear 2010. Lung and prostate cancer are the top cancer killers for menin the United States. Lung and breast cancer are the top cancer killersfor women in the United States. One in two men in the United States willbe diagnosed with cancer at some time during his lifetime. One in threewomen in the United States will be diagnosed with cancer at some timeduring her lifetime. A cure for cancer has yet to be found. Currenttreatment options, such as surgery, chemotherapy and radiationtreatment, are often times either ineffective or present serious sideeffects.

B. B Cell Malignancies

B cell malignancies, including, but not limited to, B-cell lymphomas andleukemias, are neoplastic diseases with significant incidence in theUnited States. There are approximately 55,000 new lymphoma cases of peryear in the U.S. (1998 data), with an estimated 25,000 deaths per year.This represents 4% of cancer incidence and 4% of all cancer-relateddeaths in the U.S. population. The revised European-Americanclassification of lymphoid neoplasms (1994 REAL classification, modified1999) grouped lymphomas based on their origin as either B cell lineagelymphoma, T cell lineage lymphoma, or Hodgkin's lymphoma. Lymphoma ofthe B cell lineage is the most common type of non-Hodgkin's lymphoma(NHL) diagnosed in the U.S. (Williams, HEMATOLOGY, 6^(th) ed. Beutler etal. eds., McGraw Hill, 2001).

Chronic lymphocytic leukemia (CLL) is a neoplastic disease characterizedby the accumulation of small, mature-appearing lymphocytes in the blood,marrow, and lymphoid tissues. CLL has an incidence of 2.7 cases per100,000 in the U.S. The risk increases progressively with age,particularly in men. It accounts for 0.8% of all cancers and is the mostcommon adult leukemia, responsible for 30% of all leukemias. In nearlyall cases (>98%) the diseased cells belong to the B lymphocyte lineage.A non-leukemic variant, small lymphocytic lymphoma, constitutes 5-10% ofall lymphomas, has histological, morphological and immunologicalfeatures indistinguishable from that of involved lymph nodes in patientswith B-CLL (Williams, HEMATOLOGY, 6^(th) ed. Beutler et al. eds., McGrawHill, 2001).

The natural history of chronic lymphocytic leukemia falls into severalphases. In the early phase, chronic lymphocytic leukemia is an indolentdisease, characterized by the accumulation of small, mature,functionally incompetent malignant B-cells having a lengthened lifespan. Eventually, the doubling time of the malignant B-cells decreasesand patients become increasingly symptomatic. While treatment withchemotherapeutic agents can provide symptomatic relief, the overallsurvival of the patients is only minimally extended. The late stages ofchronic lymphocytic leukemia are characterized by significant anemiaand/or thrombocytopenia. At this point, the median survival is less thantwo years (Foon et al. (1990) “Chronic Lymphocytic Leukemia: NewInsights Into Biology And Therapy,” Annals Int. Medicine 113:525-539).Due to the very low rate of cellular proliferation, chronic lymphocyticleukemia is resistant to treatment with chemotherapeutic agents.

Recently, gene expression studies have identified several genes that maybe up-regulated in lymphoproliferative disorders. One molecule thoughtto be over-expressed in patients with B-cell chronic lymphocyticleukemia (B-CLL) and in a large fraction of non-Hodgkin lymphomapatients is CD32B (Alizadeh et al. (2000) “Distinct Types Of DiffuseLarge B-Cell Lymphoma Identified By Gene Expression Profiling,” Nature403:503-511; Rosenwald et al. (2001) “Relation Of Gene ExpressionPhenotype To Immunoglobulin Mutation Genotype In B Cell ChronicLymphocytic Leukemia,” J. Exp. Med. 184:1639-1647). However, the role ofCD32B is B-CLL is unclear since one report demonstrates that CD32B wasexpressed on a low percentage of B-CLL cells and at a low density (Damleet al. (2002) “B-Cell Chronic Lymphocytic Leukemia Cells Express ASurface Membrane Phenotype Of Activated, Antigen-Experienced BLymphocytes,” Blood 99:4087-4093). CD32B is a B cell lineage surfaceantigen, whose over-expression in B cell neoplasia makes it a suitabletarget for therapeutic antibodies. In addition, CD32B belongs to thecategory of inhibitory receptors, whose ligation delivers a negativesignal. Therefore, antibodies directed against CD32B could function toeliminate tumor cells by mechanisms that include complement dependentcytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), butalso triggering an apoptotic signal. The high homology of CD32B with itscounterpart, CD32A, an activating Fcγ receptor, has thus far hamperedthe generation of antibodies that selectively recognize one but not theother form of the molecule.

Currently, cancer therapy may involve surgery, chemotherapy, hormonaltherapy and/or radiation treatment to eradicate neoplastic cells in apatient (See, for example, Stockdale, 1998, “Principles of CancerPatient Management,” in SCIENTIFIC AMERICAN MEDICINE, vol. 3, Rubensteinand Federman, eds., Chapter 12, Section IV). Recently, cancer therapycould also involve biological therapy or immunotherapy. All of theseapproaches pose significant drawbacks for the patient. Surgery, forexample, may be contraindicated due to the health of the patient or maybe unacceptable to the patient. Additionally, surgery may not completelyremove the neoplastic tissue. Radiation therapy is only effective whenthe neoplastic tissue exhibits a higher sensitivity to radiation thannormal tissue, and radiation therapy can also often elicit serious sideeffects. Hormonal therapy is rarely given as a single agent and althoughcan be effective, is often used to prevent or delay recurrence of cancerafter other treatments have removed the majority of the cancer cells.Biological therapies/immunotherapies are limited in number and mayproduce side effects such as rashes or swellings, flu-like symptoms,including fever, chills and fatigue, digestive tract problems orallergic reactions.

With respect to chemotherapy, there are a variety of chemotherapeuticagents available for treatment of cancer. A significant majority ofcancer chemotherapeutics act by inhibiting DNA synthesis, eitherdirectly or indirectly, by inhibiting the biosynthesis of thedeoxyribonucleotide triphosphate precursors, to prevent DNA replicationand concomitant cell division (See, for example, Gilman et al., GOODMANAND GILMAN'S: THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 8^(th) Ed.,Pergamom Press, New York, 1990). These agents, which include alkylatingagents, such as nitrosourea, anti-metabolites, such as methotrexate andhydroxyurea, and other agents, such as etoposides, camptothecins,bleomycin, doxorubicin, daunorubicin, etc., although not necessarilycell cycle specific, kill cells during S phase because of their effecton DNA replication. Other agents, specifically colchicine and the vincaalkaloids, such as vinblastine and vincristine, interfere withmicrotubule assembly resulting in mitotic arrest. Chemotherapy protocolsgenerally involve administration of a combination of chemotherapeuticagents to increase the efficacy of treatment.

Despite the availability of a variety of chemotherapeutic agents,chemotherapy has many drawbacks (See, for example, Stockdale, 1998,“Principles Of Cancer Patient Management,” in SCIENTIFIC AMERICANMEDICINE, vol. 3, Rubenstein and Federman, eds., ch. 12, sect. 10).Almost all chemotherapeutic agents are toxic, and chemotherapy causessignificant, and often dangerous, side effects, including severe nausea,bone marrow depression, immunosuppression, etc. Additionally, even withadministration of combinations of chemotherapeutic agents, many tumorcells are resistant or develop resistance to the chemotherapeuticagents. In fact, those cells resistant to the particularchemotherapeutic agents used in the treatment protocol often prove to beresistant to other drugs, even those agents that act by mechanismsdifferent from the mechanisms of action of the drugs used in thespecific treatment; this phenomenon is termed pleiotropic drug ormultidrug resistance. Thus, because of drug resistance, many cancersprove refractory to standard chemotherapeutic treatment protocols.

B cell malignancy is generally treated with single agent chemotherapy,combination chemotherapy and/or radiation therapy. These treatments canreduce morbidity and/or improve survival, albeit they carry significantside effects. The response of B-cell malignancies to various forms oftreatment is mixed. For example, in cases in which adequate clinicalstaging of non-Hodgkin's lymphoma is possible, field radiation therapycan provide satisfactory treatment. Certain patients, however, fail torespond and disease recurrence with resistance to treatment ensues withtime, particularly with the most aggressive variants of the disease.About one-half of the patients die from the disease (Devesa et al.(1987) “Cancer Incidence And Mortality Trends Among Whites In The UnitedStates, 1947-84,” J. Nat'l Cancer Inst. 79:701-770).

Investigational therapies for the treatment of refractory B cellneoplasia include autologous and allogeneic bone marrow or stem celltransplantation and gene therapies. Recently, immunotherapy usingmonoclonal antibodies to target B-cell specific antigens has beenintroduced in the treatment of B cell neoplasia. The use of monoclonalantibodies to direct radionuclides, toxins, or other therapeutic agentsoffers the possibility that such agents can be delivered selectively totumor sites, thus limiting toxicity to normal tissues.

There is a significant need for alternative cancer treatments,particularly for treatment of cancer that has proved refractory tostandard cancer treatments, such as surgery, radiation therapy,chemotherapy, and hormonal therapy. A promising alternative isimmunotherapy, in which cancer cells are specifically targeted by cancerantigen-specific antibodies. Major efforts have been directed atharnessing the specificity of the immune response; for example,hybridoma technology has enabled the development of tumor selectivemonoclonal antibodies (See Green M. C. et al. (2000) “MonoclonalAntibody Therapy For Solid Tumors,” Cancer Treat Rev., 26: 269-286;Weiner L M (1999) “Monoclonal Antibody Therapy Of Cancer,” Semin Oncol.26(suppl. 14):43-51), and in the past few years, the Food and DrugAdministration has approved the first MAbs for cancer therapy: RITUXAN®(rituximab) (anti-CD20) for non-Hodgkin's Lymphoma, CAMPATH®(alemtuzumab) (anti-CD52) for B-cell chronic lymphocytic leukemia(B-CLL) and HERCEPTIN® (trastuzumab) [anti-(c-erb-2/HER-2)] formetastatic breast cancer (S. A. Eccles (2001) “Monoclonal AntibodiesTargeting Cancer: ‘Magic Bullets’ Or Just The Trigger?,” Breast CancerRes., 3: 86-90). NHL and B-CLL are two of the most common forms of Bcell neoplasia. These antibodies have demonstrated clinical efficacy,but their use is not without side effects. The potency of antibodyeffector function, e.g., to mediate antibody dependent cellularcytotoxicity (“ADCC”) is an obstacle to such treatment. Furthermore,with RITUXAN® (rituximab) and CAMPATH® (alemtuzumab), at least half thepatients fail to respond and a fraction of responders may be refractoryto subsequent treatments.

There is a need for alternative therapies for cancer, particularly,B-cell malignancies, especially for patients that are refractory forstandard cancer treatments and new immunotherapies such as RITUXAN®(rituximab).

C. Inflammatory Diseases and Autoimmune Diseases

Inflammation is a process by which the body's white blood cells andchemicals protect our bodies from infection by foreign substances, suchas bacteria and viruses. It is usually characterized by pain, swelling,warmth and redness of the affected area. Chemicals known as cytokinesand prostaglandins control this process, and are released in an orderedand self-limiting cascade into the blood or affected tissues. Thisrelease of chemicals increases the blood flow to the area of injury orinfection, and may result in the redness and warmth. Some of thechemicals cause a leak of fluid into the tissues, resulting in swelling.This protective process may stimulate nerves and cause pain. Thesechanges, when occurring for a limited period in the relevant area, workto the benefit of the body.

In autoimmune and/or inflammatory disorders, the immune system triggersan inflammatory response when there are no foreign substances to fightand the body's normally protective immune system causes damage to itsown tissues by mistakenly attacking self. There are many differentautoimmune disorders that affect the body in different ways. Forexample, the brain is affected in individuals with multiple sclerosis,the gut is affected in individuals with Crohn's disease, and thesynovium, bone and cartilage of various joints are affected inindividuals with rheumatoid arthritis. As autoimmune disorders progressdestruction of one or more types of body tissues, abnormal growth of anorgan, or changes in organ function may result. The autoimmune disordermay affect only one organ or tissue type or may affect multiple organsand tissues. Organs and tissues commonly affected by autoimmunedisorders include red blood cells, blood vessels, connective tissues,endocrine glands (e.g., the thyroid or pancreas), muscles, joints, andskin. Examples of autoimmune disorders include, but are not limited to,Hashimoto's thyroiditis, pernicious anemia, Addison's disease, type 1diabetes, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, dermatomyositis, lupuserythematosus, multiple sclerosis, autoimmune inner ear diseasemyasthenia gravis, Reiter's syndrome, Graves disease, autoimmunehepatitis, familial adenomatous polyposis and ulcerative colitis.

Rheumatoid arthritis (RA) and juvenile rheumatoid arthritis are types ofinflammatory arthritis. Arthritis is a general term that describesinflammation in joints. Some, but not all, types of arthritis are theresult of misdirected inflammation. Besides rheumatoid arthritis, othertypes of arthritis associated with inflammation include the following:psoriatic arthritis, Reiter's syndrome, ankylosing spondylitisarthritis, and gouty arthritis. Rheumatoid arthritis is a type ofchronic arthritis that occurs in joints on both sides of the body (suchas both hands, wrists or knees). This symmetry helps distinguishrheumatoid arthritis from other types of arthritis. In addition toaffecting the joints, rheumatoid arthritis may occasionally affect theskin, eyes, lungs, heart, blood or nerves.

Rheumatoid arthritis affects about 1% of the world's population and ispotentially disabling. There are approximately 2.9 million incidences ofrheumatoid arthritis in the United States. Two to three times more womenare affected than men. The typical age that rheumatoid arthritis occursis between 25 and 50. Juvenile rheumatoid arthritis affects 71,000 youngAmericans (aged eighteen and under), affecting six times as many girlsas boys.

Rheumatoid arthritis is an autoimmune disorder where the body's immunesystem improperly identifies the synovial membranes that secrete thelubricating fluid in the joints as foreign. Inflammation results, andthe cartilage and tissues in and around the joints are damaged ordestroyed. In severe cases, this inflammation extends to other jointtissues and surrounding cartilage, where it may erode or destroy boneand cartilage and lead to joint deformities. The body replaces damagedtissue with scar tissue, causing the normal spaces within the joints tobecome narrow and the bones to fuse together. Rheumatoid arthritiscreates stiffness, swelling, fatigue, anemia, weight loss, fever, andoften, crippling pain. Some common symptoms of rheumatoid arthritisinclude joint stiffness upon awakening that lasts an hour or longer;swelling in a specific finger or wrist joints; swelling in the softtissue around the joints; and swelling on both sides of the joint.Swelling can occur with or without pain, and can worsen progressively orremain the same for years before progressing.

The diagnosis of rheumatoid arthritis is based on a combination offactors, including: the specific location and symmetry of painfuljoints, the presence of joint stiffness in the morning, the presence ofbumps and nodules under the skin (rheumatoid nodules), results of X-raytests that suggest rheumatoid arthritis, and/or positive results of ablood test called the rheumatoid factor. Many, but not all, people withrheumatoid arthritis have the rheumatoid-factor antibody in their blood.The rheumatoid factor may be present in people who do not haverheumatoid arthritis. Other diseases can also cause the rheumatoidfactor to be produced in the blood. That is why the diagnosis ofrheumatoid arthritis is based on a combination of several factors andnot just the presence of the rheumatoid factor in the blood.

The typical course of the disease is one of persistent but fluctuatingjoint symptoms, and after about 10 years, 90% of sufferers will showstructural damage to bone and cartilage. A small percentage will have ashort illness that clears up completely, and another small percentagewill have very severe disease with many joint deformities, andoccasionally other manifestations of the disease. The inflammatoryprocess causes erosion or destruction of bone and cartilage in thejoints. In rheumatoid arthritis, there is an autoimmune cycle ofpersistent antigen presentation, T-cell stimulation, cytokine secretion,synovial cell activation, and joint destruction. The disease has a majorimpact on both the individual and society, causing significant pain,impaired function and disability, as well as costing millions of dollarsin healthcare expenses and lost wages.

Currently available therapy for arthritis focuses on reducinginflammation of the joints with anti-inflammatory or immunosuppressivemedications. The first line of treatment of any arthritis is usuallyanti-inflammatories, such as aspirin, ibuprofen and Cox-2 inhibitorssuch as celecoxib and rofecoxib. “Second line drugs” include gold,methotrexate and steroids. Although these are well-establishedtreatments for arthritis, very few patients remit on these lines oftreatment alone. Recent advances in the understanding of thepathogenesis of rheumatoid arthritis have led to the use of methotrexatein combination with antibodies to cytokines or recombinant solublereceptors. For example, recombinant soluble receptors for tumor necrosisfactor (TNF)-α have been used in combination with methotrexate in thetreatment of arthritis. However, only about 50% of the patients treatedwith a combination of methotrexate and anti-TNF-α agents such asrecombinant soluble receptors for TNF-α show clinically significantimprovement. Many patients remain refractory despite treatment.Difficult treatment issues still remain for patients with rheumatoidarthritis. Many current treatments have a high incidence of side effectsor cannot completely prevent disease progression. So far, no treatmentis ideal, and there is no cure. Novel therapeutics are needed that moreeffectively treat rheumatoid arthritis and other autoimmune disorders.

D. Allergy

Immune-mediated allergic (hypersensitivity) reactions are classifiedinto four types (I-IV) according to the underlying mechanisms leading tothe expression of the allergic symptoms. Type I allergic reactions arecharacterized by IgE-mediated release of vasoactive substances such ashistamine from mast cells and basophils. The release of these substancesand the subsequent manifestation of allergic symptoms are initiated bythe cross-linking of allergen-bound IgE to its receptor on the surfaceof mast cells and basophils. In individuals suffering from type Iallergic reactions, exposure to an allergen for a second time leads tothe production of high levels of IgE antibodies specific for theallergen as a result of the involvement of memory B and T cells in the3-cell interaction required for IgE production. The high levels of IgEantibodies produced cause an increase in the cross-linking of IgEreceptors on mast cells and basophils by allergen-bound IgE, which inturn leads to the activation of these cells and the release of thepharmacological mediators that are responsible for the clinicalmanifestations of type I allergic diseases.

Two receptors with differing affinities for IgE have been identified andcharacterized. The high affinity receptor (FcεRI) is expressed on thesurface of mast cells and basophils. The low affinity receptor(FcεRII/CD23) is expressed on many cell types including B cells, Tcells, macrophages, eosinophils and Langerhan cells. The high affinityIgE receptor consists of three subunits (alpha, beta and gamma chains).Several studies demonstrate that only the alpha chain is involved in thebinding of IgE, whereas the beta and gamma chains (which are eithertransmembrane or cytoplasmic proteins) are required for signaltransduction events. The identification of IgE structures required forIgE to bind to the FcεRI on mast cells and basophils is of utmostimportance in devising strategies for treatment or prevention ofIgE-mediated allergies. For example, the elucidation of the IgEreceptor-binding site could lead to the identification of peptides orsmall molecules that block the binding of IgE to receptor-bearing cellsin vivo.

Currently, IgE-mediated allergic reactions are treated with drugs suchas antihistamines and corticosteroids which attempt to alleviate thesymptoms associated with allergic reactions by counteracting the effectsof the vasoactive substances released from mast cells and basophils.High doses of antihistamines and corticosteroids have deleterious sideeffects (e.g., central nervous system disturbance, constipation, etc).Thus, other methods for treating type I allergic reactions are needed.

One approach to the treatment of type I allergic disorders has been theproduction of monoclonal antibodies which react with soluble (free) IgEin serum, block IgE from binding to its receptor on mast cells andbasophils, and do not bind to receptor-bound IgE (i.e., they arenon-anaphylactogenic). Two such monoclonal antibodies are in advancedstages of clinical development for treatment of IgE-mediated allergicreactions (see, e.g., Chang, T. W. (2000) “The Pharmacological Basis OfAnti-IgE Therapy,” Nature Biotechnology 18:157-162).

One of the most promising treatments for IgE-mediated allergic reactionsis the active immunization against appropriate non-anaphylactogenicepitopes on endogenous IgE. Stanworth et al. (U.S. Pat. No. 5,601,821)described a strategy involving the use of a peptide derived from theCεH4 domain of the human IgE coupled to a heterologous carrier proteinas an allergy vaccine. However, this peptide has been shown not toinduce the production of antibodies that react with native soluble IgE.Further, Hellman (U.S. Pat. No. 5,653,980) proposed anti-IgE vaccinecompositions based on fusion of full length CεH2-CεH3 domains(approximately 220 amino acid long) to a foreign carrier protein.However, the antibodies induced by the anti-IgE vaccine compositionsproposed in Hellman will most likely it result in anaphylaxis, sinceantibodies against some portions of the CεH2 and CεH3 domains of the IgEmolecule have been shown to cross-link the IgE receptor on the surfaceof mast cell and basophils and lead to production of mediators ofanaphylaxis (See, e.g., Stadler et al. (1993) “Biological Activities OfAnti-IgE Antibodies,” Int. Arch. Allergy and Immunology 102: 121-126).Therefore, a need remains for treatment of IgE-mediated allergicreactions which do not induce anaphylactic antibodies.

The significant concern over induction of anaphylaxis has resulted inthe development of another approach to the treatment of type I allergicdisorders consisting of mimotopes that could induce the production ofanti-IgE polyclonal antibodies when administered to animals (See, e.g.,Rudolf, et al. (1998) “Epitope-Specific Antibody Response To IgE ByMimotope Immunization,” Journal of Immunology 160:3315-3321). Kricek etal. (International Publication No. WO 97/31948) screened phage-displayedpeptide libraries with the monoclonal antibody BSWI7 to identify peptidemimotopes that could mimic the conformation of the IgE receptor binding.These mimotopes could presumably be used to induce polyclonal antibodiesthat react with free native IgE, but not with receptor-bound IgE as wellas block IgE from binding to its receptor. Kricek et al. disclosedpeptide mimotopes that are not homologous to any part of the IgEmolecule and are thus different from peptides disclosed in the presentinvention.

As evidenced by a survey of the art, there remains a need for enhancingthe therapeutic efficacy of current methods of treating or preventingdisorders such as cancer, autoimmune disease, inflammatory disorder, orallergy. In particular, there is a need for enhancing the effectorfunction, particularly, the cytotoxic effect of therapeutic antibodiesused in treatment of cancer. The current state of the art is alsolacking in treating or preventing allergy disorders (e.g., either byantibody therapy or vaccine therapy).

SUMMARY OF THE INVENTION

The instant invention provides humanized FcγRIIB antibodies, an isolatedantibody or a fragment thereof that specifically binds FcγRIIB,particularly human FcγRIIB, more particularly native human FcγRIIB, witha greater affinity than said antibody or a fragment thereof bindsFcγRIIA, particularly human FcγRIIA, more particularly native humanFcγRIIA. As used herein, “native FcγRIIB or FcγRIIA” means FcγRIIB orFcγRIIA which is endogenously expressed in a cell and is present on thecell surface of that cell or recombinantly expressed in a mammalian celland present on the cell surface, but is not FcγRIIB or FcγRIIA expressedin a bacterial cell or denatured, isolated FcγRIIB or FcγRIIA. Theinstant invention encompasses humanized antibodies, and antigen bindingfragments thereof, derived from antibodies that bind FcγRIIB,particularly human FcγRIIB, more particularly native human FcγRIIB, witha greater affinity than said antibody or a fragment thereof bindsFcγRIIA, particularly human FcγRIIA, more particularly native humanFcγRIIA. In most preferred embodiments, the instant invention relates tohumanized 2B6 or 3H7 antibodies or fragments thereof, preferably antigenbinding fragments thereof. In another preferred embodiments, theinvention relates to humanized 1D5, 2E1, 2H9, 2D11, or 1F2 antibodiesand fragments thereof, preferably antigen binding fragments thereof.

Preferably the humanized antibodies of the invention bind theextracellular domain of native human FcγRIIB. The humanized anti-FcγRIIBantibodies of the invention may have a heavy chain variable regioncomprising a CDR1 having the amino acid sequence of NYWIH (SEQ ID NO:1)or DAWMD (SEQ ID NO:29) and/or a CDR2 having the amino acid sequence ofVIDPSDTYPNYNKKFK (SEQ ID NO:2) or EIRNKANNLATYYAES (SEQ ID NO:30) and/ora CDR3 having the amino acid sequence of NGDSDYYSGMDY (SEQ ID NO:3) orYSPFAY (SEQ ID NO:31) and/or a light chain variable region comprising aCDR1 having the amino acid sequence of RTSQSIGTNIH (SEQ ID NO:8) orRASQEISGYLS (SEQ ID NO:38) and/or a CDR2 having the amino acid sequenceof NVSESIS (SEQ ID NO:9), YVSESIS (SEQ ID NO:10), YASESIS (SEQ IDNO:11), or AASTLDS (SEQ ID NO:39) and/or a CDR3 having the amino acidsequence of QQSNTWPFT (SEQ ID NO:12) or LQYVSYPYT (SEQ ID NO:40).

In yet other preferred embodiments, the humanized antibodies of theinvention comprise a light chain variable region comprising an aminoacid sequence of:

(SEQ ID NO:18) EIVLTQSPDFQSVTPKEKVTITCRTSQSIGTNIHWYQQKPDQSPKLLIKNVSESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNTWPFTF GGGTKVEIK; ora light chain variable region comprising an amino acid sequence of:

(SEQ ID NO:20) EIVLTQSPDFQSVTPKEKVTITCRTSQSIGTNIHWYQQKPDQSPKLLIKYVSESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNTWPFTF GGGTKVEIK; ora light chain variable region comprising an amino acid sequence of:

(SEQ ID NO:22) EIVLTQSPDFQSVTPKEKVTITCRTSQSIGTNIHWYQQKPDQSPKLLIKYASESISGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNTWPFTF GGGTKVEIK; ora light chain variable region comprising an amino acid sequence of:

(SEQ ID NO:46) DIQMTQSPSSLSASLGERVSLTCRASQEISGYLSWLQQKPDGTIRRLIYAASTLDSGVPKRFSGSWSGSDYSLTISSLESEDFADYYCLQYVSYPYTF GGGTKLEIK; and/ora heavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO:24) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGVIDPSDTYPNYNKKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARNGDS DYYSGMDYWGQGTTVTVSS;ora heavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO:37) EVKFEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQGPEKGLEWVAEIRNKANNLATYYAESVKGRFTIPRDDSKSSVYLHMNSLRAEDTGIY YCYSPFAYWGQGTLVTVSA;and/or amino acid sequence variants thereof.

In particular, the invention provides a humanized antibody thatimmunospecifically binds to extracellular domain of native humanFcγRIIB, said antibody comprising (or alternatively, consisting of) a VHCDR1 and a VL CDR1; a VH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; aVH CDR2 and a VL CDR1; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; aVH CDR3 and a VH CDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3;a VH1 CDR1, a VH CDR2 and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2;a VH CDR1, a VH CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1,a VH CDR2, a VH CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3;a VH CDR1, a VL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3;a VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3;a VH CDR3, a VL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL CDR3;a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, aVH CDR3 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; aVH CDR1, a VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VHCDR1, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VLCDR3; a VH CDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combinationthereof of the VH CDRs and VL CDRs disclosed herein.

In one specific embodiment, the invention provides a humanized 2B6antibody, wherein the VH region consists of the FR segments from thehuman germline VH segment VH1-18 and JH6, and the CDR regions of the 2B6VH, having the amino acid sequence of SEQ ID NO:24. In another specificembodiment, the humanized 2B6 antibody further comprises a VL regions,which consists of the FR segments of the human germline VL segmentVK-A26 and JK4 and the CDR regions of 2B6VL, having an amino acidsequence of SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22.

In one specific embodiment, the invention provides a humanized 3H7antibody, wherein the VH region consists of the FR segments from a humangermline VH segment, and the CDR regions of the 3H7 VH, having the aminoacid sequence of SEQ ID NO:37. In another specific embodiment, thehumanized 3H7 antibody further comprises a VL regions, which consists ofthe FR segments of the human germline VL segment, and the CDR regions of3H7VL, having an amino acid sequence of SEQ ID NO:46.

The present invention provides humanized antibody molecules specific forFcγRIIB in which one or more regions of one or more CDRs of the heavyand/or light chain variable regions of a human antibody (the recipientantibody) have been substituted by analogous parts of one or more CDRsof a donor monoclonal antibody which specifically binds FcγRIIB, with agreater affinity than FcγRIIA, e.g., monoclonal antibody produced byclones 2B6 and 3H7 which bind FcγRIIB, having ATCC accession numbersPTA-4591, and PTA-4592, respectively, or a monoclonal antibody producedby clones 1D5, 2E1, 2H9, 2D11, and 1F2 having ATCC Accession numbers,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively. In amost preferred embodiment, the humanized antibody can specifically bindto the same epitope as the donor murine antibody. It will be appreciatedby one skilled in the art that the invention encompasses CDR grafting ofantibodies in general. Thus, the donor and acceptor antibodies may bederived from animals of the same species and even same antibody class orsub-class. More usually, however, the donor and acceptor antibodies arederived from animals of different species. Typically the donor antibodyis a non-human antibody, such as a rodent MAb, and the acceptor antibodyis a human antibody.

In some embodiments, at least one CDR from the donor antibody is graftedonto the human antibody. In other embodiments, at least two andpreferably all three CDRs of each of the heavy and/or light chainvariable regions are grafted onto the human antibody. The CDRs maycomprise the Kabat CDRs, the structural loop CDRs or a combinationthereof. In some embodiments, the invention encompasses a humanizedFcγRIIB antibody comprising at least one CDR grafted heavy chain and atleast one CDR-grafted light chain.

In a preferred embodiment, the CDR regions of the humanizedFcγRIIB-specific antibody are derived from the murine antibody againstFcγRIIB. In some embodiments, the humanized antibodies described hereincomprise alterations, including, but not limited to, amino aciddeletions, insertions, and modifications, of the acceptor antibody,i.e., human, heavy and/or light chain variable domain framework regionsthat are necessary for retaining binding specificity of the donormonoclonal antibody. In some embodiments, the framework regions of thehumanized antibodies described herein do not necessarily consist of theprecise amino acid sequence of the framework region of a naturaloccurring human antibody variable region, but contain variousalterations, including, but not limited to, amino acid deletions,insertions, modifications that alter the property of the humanizedantibody, for example, improve the binding properties of a humanizedantibody region that is specific for the same target as the murineFcγRIIB-specific antibody. In most preferred embodiments, a minimalnumber of alterations are made to the framework region in order to avoidlarge-scale introductions of non-human framework residues and to ensureminimal immunogenicity of the humanized antibody in humans. In someembodiments, the framework residues are derived from the human germlineVH segment VH1-18 and JH6 and/or the human germline VL segment VK-A26and JK4. In most preferred embodiments of the invention, there are noalterations made to the framework regions. The donor monoclonal antibodyof the present invention is preferably a monoclonal antibody produced byclones 2B6 and 3H7 which bind FcγRIIB, having ATCC accession numbersPTA-4591, and PTA-4592, or a monoclonal antibody produced by clones 1D5,2E1, 2H9, 2D11, and 1F2 having ATCC Accession numbers, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively.

The humanized antibodies of the present invention include completeantibody molecules having full length heavy and light chains, or anyfragment thereof, such as the Fab or (Fab′)₂ fragments, a heavy chainand light chain dimer, or any minimal fragment thereof such as an Fv, anSCA (single chain antibody), and the like, specific for the FcγRIIB.

The invention encompasses methods for the production of antibodies ofthe invention or fragments thereof, particularly for the production ofhumanized anti-FcγRIIB specific antibodies, such that the FcγRIIBspecific antibodies have an enhanced specificity for FcγRIIB relative toFcγRIIA. The invention encompasses any method known in the art usefulfor the production of polypeptides, e.g., in vitro synthesis,recombinant DNA production, and the like. Preferably, the humanizedantibodies are produced by recombinant DNA technology. The humanizedFcγRIIB specific antibodies of the invention may be produced usingrecombinant immunoglobulin expression technology. Exemplary methods forthe production of recombinant humanized antibodies of the invention maycomprise the following: (A) constructing, by conventional molecularbiology methods, an expression vector comprising an operon that encodesan antibody heavy chain in which the CDRs and a minimal portion of thevariable region framework that are required to retain donor antibodybinding specificity are derived from a non-human immunoglobulin, such asthe murine FcγRIIB specific monoclonal antibody, e.g., monoclonalantibody produced by clones 2B6 and 3H7 which bind FcγRIIB, having ATCCaccession numbers PTA-4591, and PTA-4592, respectively, or a monoclonalantibody produced by clones 1D5, 2E1, 2H9, 2D11, and 1F2 having ATCCAccession numbers, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively, and the remainder of the antibody is derived from a humanimmunoglobulin, thereby producing a vector for the expression of ahumanized antibody heavy chain; (B) constructing, by conventionalmolecular biology methods, an expression vector comprising an operonthat encodes an antibody light chain in which the CDRs and a minimalportion of the variable region framework that are required to retaindonor antibody binding specificity are derived from a non-humanimmunoglobulin, such as the murine FcγRIIB monoclonal antibody, e.g.,monoclonal antibody produced by clones 2B6 and 3H7 which bind FcγRIIB,having ATCC accession numbers PTA-4591, and PTA-4592, respectively, or amonoclonal antibody produced by clones 1D5, 2E1, 2H9, 2D11, and 1F2having ATCC Accession numbers, PTA-5958, PTA-5961, PTA-5962, PTA-5960,and PTA-5959, respectively, and the remainder of the antibody is derivedfrom a human immunoglobulin, thereby producing a vector for theexpression of humanized antibody light chain; (C) transferring theexpression vectors to a host cell by conventional molecular biologymethods to produce a transfected host cell for the expression ofhumanized anti-FcγRIIB antibodies; and (D) culturing the transfectedcell by conventional cell culture techniques so as to produce humanizedanti-FcγRIIB antibodies. Host cells may be cotransfected with twoexpression vectors of the invention, the first vector containing anoperon encoding a heavy chain derived polypeptide and the secondcontaining an operon encoding a light chain derived polypeptide. The twovectors may contain different selectable markers but, with the exceptionof the heavy and light chain coding sequences, are preferably identical.This procedure provides for equal expression of heavy and light chainpolypeptides. Alternatively, a single vector may be used which encodesboth heavy and light chain polypeptides. The coding sequences for theheavy and light chains may comprise cDNA or genomic DNA or both. Thehost cell used to express the recombinant antibody of the invention maybe either a bacterial cell such as Escherichia coli, or, preferably, aeukaryotic cell. Preferably, a mammalian cell such as a chinese hamsterovary cell or HEK-293 may be used. The choice of expression vector isdependent upon the choice of host cell, and may be selected so as tohave the desired expression and regulatory characteristics in theselected host cell. The general methods for construction of the vectorof the invention, transfection of cells to produce the host cell of theinvention, culture of cells to produce the antibody of the invention areall conventional molecular biology methods. Likewise, once produced, therecombinant antibodies of the invention may be purified by standardprocedures of the art, including cross-flow filtration, ammoniumsulphate precipitation, affinity column chromatography, gelelectrophoresis and the like.

In some embodiments, cell fusion methods for making monoclonalantibodies may be used in the methods of the invention such as thosedisclosed in U.S. Pat. No. 5,916,771, incorporated herein by referencein its entirety. Briefly, according to this method, DNA encoding thedesired heavy chain (or a fragment of the heavy chain) is introducedinto a first mammalian host cell, while DNA encoding the desired lightchain (or a fragment of the light chain) is introduced into a secondmammalian host cell. The first transformed host cell and the secondtransformed host cell are then combined by cell fusion to form a thirdcell. Prior to fusion of the first and second cells, the transformedcells may be selected for specifically desired characteristics, e.g.,high levels of expression. After fusion, the resulting hybrid cellcontains and expresses both the DNA encoding the desired heavy chain andthe DNA encoding the desired light chain, resulting in production of themultimeric antibody.

The invention encompasses using the humanized antibodies of the presentinvention in conjunction with, or attached to, other antibodies orfragments thereof such as human or humanized monoclonal antibodies.These other antibodies may be reactive with other markers (epitopes)characteristic for the disease against which the antibodies of theinvention are directed or may have different specificities chosen, forexample, to recruit molecules or cells of the human immune system to thediseased cells. The antibodies of the invention (or parts thereof) maybe administered with such antibodies (or parts thereof) as separatelyadministered compositions or as a single composition with the two agentslinked by conventional chemical or by molecular biological methods.Additionally the diagnostic and therapeutic value of the antibodies ofthe invention may be augmented by labelling the humanized antibodieswith labels that produce a detectable signal (either in vitro or invivo) or with a label having a therapeutic property. Some labels, e.g.,radionucleotides may produce a detectable signal and have a therapeuticproperty. Examples of radionuclide labels include ¹²⁵I, ¹³¹I, ¹⁴C.Examples of other detectable labels include a fluorescent chromophoresuch as fluorescein, phycobiliprotein or tetraethyl rhodamine forfluorescence microscopy; an enzyme which produces a fluorescent orcolored product for detection by fluorescence, absorbance, visible coloror agglutination, or which produces an electron dense product fordemonstration by electron microscopy; or an electron dense molecule suchas ferritin, peroxidase or gold beads for direct or indirect electronmicroscopic visualization. Labels having therapeutic properties includedrugs for the treatment of cancer, such as methotrexate and the like.

The methods of the invention also encompass polynucleotides that encodethe humanized antibodies of the invention. In one embodiment, theinvention provides an isolated nucleic acid sequence encoding a heavychain or a light chain of an antibody or a fragment thereof thatspecifically binds FcγRIIB with greater affinity than said antibody or afragment thereof binds FcγRIIA. The invention also relates to a vectorcomprising said nucleic acid. The invention further provides a vectorcomprising a first nucleic acid molecule encoding a heavy chain and asecond nucleic acid molecule encoding a light chain, said heavy chainand light chain being of an antibody or a fragment thereof thatspecifically binds FcγRIIB with greater affinity than said antibody or afragment thereof binds FcγRIIA. In one specific embodiment, said vectoris an expression vector. The invention further provides host cellscontaining the vectors or polynucleotides encoding the antibodies of theinvention. Preferably, the invention encompasses polynucleotidesencoding heavy and light chains of the antibodies produced by thedeposited hybridoma clones, having ATCC accession numbers PTA-4591 andPTA-4592, respectively, or ATCC Accession numbers, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively, or portions thereof,e.g., CDRs, variable domains, etc. and humanized versions thereof.

The invention encompasses the use of the humanized antibodies of theinvention to detect the presence of FcγRIIB specifically (i.e., FcγRIIBand not FcγRIIA) in a biological sample.

Activating and inhibitory Fc receptors, e.g., FcγRIIA and FcγRIIB, arecritical for the balanced function of these receptors and propercellular immune responses. The invention encompasses the use of thehumanized antibodies of the invention for the treatment of any diseaserelated to loss of such balance and regulated control in the Fc receptorsignaling pathway. Thus, the humanized FcγRIIB antibodies of theinvention have uses in regulating the immune response, e.g., ininhibiting immune response in connection with autoimmune or inflammatorydisease, or allergic response. The humanized FcγRIIB antibodies of theinvention can also be used to alter certain effector functions toenhance, for example, therapeutic antibody-mediated cytotoxicity.

The humanized antibodies of the invention are useful for prevention ortreatment of cancer, for example, in one embodiment, as a single agenttherapy. In one embodiment of the invention, the humanized antibodies ofthe invention are useful for prevention or treatment of B-cellmalignancies, particularly non-Hodgkin's lymphoma or chronic lymphocyticleukemia. In particular embodiments, the cancer of the subject isrefractory to one or more standard or experimental therapies,particularly, to RITUXAN® (rituximab) treatment. The methods of theinvention may be used for the treatment, management, prevention, oramelioration of B-cell diseases, such as, B-cell chronic lymphocyticleukemia (B-CLL), non-Hodgkin's lymphoma, diffuse large B cell lymphoma,follicular lymphoma with areas of diffuse large B cell lymphoma, smalllymphocytic lymphoma, mantle cell lymphoma, and diffuse small cleavedcell lymphoma.

In another embodiment, the invention provides for the use of aFcγRIIB-specific antibody conjugated to a therapeutic agent or drug.Examples of therapeutic agents which may be conjugated to ananti-FcγRIIB antibody or an antigen-binding fragment thereof include,but are not limited to, cytokines, toxins, radioactive elements, andantimetabolites.

In one embodiment, the invention provides for the use of a humanizedFcγRIIB-specific antibody in combination with a standard or experimentaltreatment regimen for B-cell malignancies (e.g., chemotherapy,radioimmunotherapy, or radiotherapy). Such combination therapy mayenhance the efficacy of standard or experimental treatment. Examples oftherapeutic agents that are particularly useful in combination with aFcγRIIB-specific antibody or an antigen-binding fragment thereof, forthe prevention, treatment, management, or amelioration of B-cellmalignancies, include, but are not limited to, RITUXAN® (rituximab),interferon-alpha, and anti-cancer agents. Chemotherapeutic agents thatcan be used in combination with a FcγRIIB-specific antibody or anantigen-binding fragment thereof, include, but are not limited toalkylating agents, antimetabolites, natural products, and hormones. Thecombination therapies of the invention enable lower dosages of ananti-FcγRIIB antibody or an antigen-binding fragment thereof and/or lessfrequent administration of anti-FcγRIIB antibody or an antigen-bindingfragment thereof to a subject with a B-cell malignancy, to achieve atherapeutic or prophylactic effect.

In another embodiment, the use of a humanized FcγRIIB antibody or anantigen-binding fragment thereof prolongs the survival of a subjectdiagnosed with a B-cell malignancy.

In a preferred embodiment, the humanized antibodies of the invention areused for the treatment and/or prevention of melanoma. In anotherembodiment, the humanized antibodies are useful for prevention and/ortreatment of cancer, particularly in potentiating the cytotoxic activityof cancer antigen-specific therapeutic antibodies with cytotoxicactivity to enhance tumor cell killing and/or enhancing antibodydependent cytotoxic cellular (“ADCC”) activity, complement dependentcytotoxic (“CDC”) activity, or phagocytosis of the therapeuticantibodies.

The invention provides a method of treating cancer in a patient having acancer characterized by a cancer antigen, said method comprisingadministering to said patient a therapeutically effective amount of afirst humanized antibody or a fragment thereof that specifically bindsFcγRIIB with greater affinity than said antibody or a fragment thereofbinds FcγRIIA, and a second antibody that specifically binds said cancerantigen and is cytotoxic. The invention also provides a method oftreating cancer in a patient having a cancer characterized by a cancerantigen, said method comprising administering to said patient atherapeutically effective amount of an humanized antibody or a fragmentthereof that specifically binds FcγRIIB, particularly native humanFcγRIIB, with greater affinity than said antibody or a fragment thereofbinds FcγRIIA, preferably native human FcγRIIA, and the constant domainof which further has an increased affinity for one or more Fc activationreceptors, when the antibody is monomeric, such as FcγRIIIA, and anantibody that specifically binds said cancer antigen and is cytotoxic.In one particular embodiment, said Fc activation receptor is FcγRIIIA.

In some embodiments, the invention encompasses antibodies comprisingvariant Fc regions that bind FcRn with an enhanced affinity, resultingin an increased antibody half life, e.g., a half-life of greater than 15days, preferably greater than 20 days, greater than 25 days, greaterthan 30 days, greater than 35 days, greater than 40 days, greater than45 days, greater than 2 months, greater than 3 months, greater than 4months, or greater than 5 months. Although not intending to be bound bya particular mechanism of action the neonatal Fc receptor (FcRn) playsan important role in regulating the serum half-lives of IgG antibodies.A correlation has been established between the pH-dependent bindingaffinity of IgG antibodies to FcRn and their serum half-lives in mice.The increased half-lives of the antibodies of the present invention orfragments thereof in a mammal, preferably a human, results in a higherserum titer of said antibodies or antibody fragments in the mammal, andthus, reduces the frequency of the administration of said antibodies orantibody fragments and/or reduces the concentration of said antibodiesor antibody fragments to be administered. For example, antibodies orfragments thereof with increased in vivo half-lives can be generated bymodifying (e.g., substituting, deleting or adding) amino acid residuesidentified as involved in the interaction between the Fc domain and theFcRn receptor. For example, the invention encompasses antibodiescomprising variant Fc regions which have at least one or moremodification that enhances the affinity to FcRn, e.g., a modification ofone or more amino acid residues 251-256, 285-290, 308-314, 385-389, and428-436, or a modification at positions 250 and 428 (see Hinton et al.(2004) “Engineered Human IgG Antibodies with Longer Serum Half-lives inPrimates,” J. Biol. Chem. 279(8):6213-6216; PCT Publication No. WO97/34631; and WO 02/060919, all of which are incorporated herein byreference in its entirety.

In another embodiment, the invention provides a method of enhancing anantibody mediated cytotoxic effect in a subject being treated with acytotoxic antibody, said method comprising administering to said patienta humanized antibody of the invention, or a fragment thereof, in anamount sufficient to enhance the cytotoxic effect of said cytotoxicantibody. In yet another embodiment, the invention provides a method ofenhancing an antibody-mediated cytotoxic effect in a subject beingtreated with a cytotoxic antibody, said method comprising administeringto said patient a humanized antibody of the invention, or a fragmentthereof, further having an enhanced affinity for an Fc inhibitoryreceptor, when monomeric, in an amount sufficient to enhance thecytotoxic effect of said cytotoxic antibody. In yet another embodiment,the invention provides a method further comprising the administration ofone or more additional cancer therapies.

The invention encompasses the use of the humanized antibodies of theinvention in combination with any therapeutic antibody that mediates itstherapeutic effect through cell killing to potentiate the antibody'stherapeutic activity. In one particular embodiment, the humanizedantibodies of the invention potentiate the antibody's therapeuticactivity by enhancing antibody-mediated effector function. In anotherembodiment of the invention, the humanized antibodies of the inventionpotentiate the cytotoxic antibody's therapeutic activity by enhancingphagocytosis and opsonization of the targeted tumor cells. In yetanother embodiment of the invention, the humanized antibodies of theinvention potentiate the antibody's therapeutic activity by enhancingantibody-dependent cell-mediated cytotoxicity (“ADCC”) in destruction ofthe targeted tumor cells. In certain embodiments, the antibodies of theinvention are used in combination with Fc fusion proteins to enhanceADCC.

Although not intending to be bound by a particular mechanism of action,the combination of a humanized antibody of the invention in combinationwith a therapeutic antibody has an enhanced therapeutic effect due, inpart, to the cytotoxic ability of the FcγRIIB specific humanizedantibody to eliminate macrophages expressing the inhibitory FcgRIIBreceptors. Therefore, there is a higher concentration of cellsexpressing activating FcgR receptors remaining per dose of thetherapeutic antibody.

In some embodiments, the invention encompasses use of the humanizedantibodies of the invention in combination with a therapeutic antibodythat does not mediate its therapeutic effect through cell killing topotentiate the antibody's therapeutic activity. In a specificembodiment, the invention encompasses use of the humanized antibodies ofthe invention in combination with a therapeutic apoptosis inducingantibody with agonistic activity, e.g., anti-Fas antibody. Therapeuticapoptosis inducing antibodies may be specific for any death receptorknown in the art for the modulation of apoptotic pathway, e.g., TNFRreceptor family member or a TRAIL family member.

The invention encompasses using the humanized antibodies of theinvention to block macrophage mediated tumor cell progression andmetastasis. The humanized antibodies of the invention are particularlyuseful in the treatment of solid tumors, where macrophage infiltrationoccurs. The antagonistic humanized antibodies of the invention areparticularly useful for controlling, e.g., reducing or eliminating,tumor cell metastasis, by reducing or eliminating the population ofmacrophages that are localized at the tumor site. The invention furtherencompasses humanized antibodies that effectively deplete or eliminateimmune effector cells other than macrophages that express FcγRIIB, e.g.,dendritic cells. Effective depletion or elimination of immune effectorcells using the antibodies of the invention may range from a reductionin population of the effector cells by 50%, 60%, 70%, 80%, preferably90%, and most preferably 99%.

In some embodiments, the invention encompasses use of the humanizedantibodies of the invention in combination with therapeutic antibodiesthat immunospecifically bind to tumor antigens that are not expressed onthe tumor cells themselves, but rather on the surrounding reactive andtumor supporting, non-malignant cells comprising the tumor stroma. In apreferred embodiment, a humanized antibody of the invention is used incombination with an antibody that immunospecifically binds a tumorantigen on a fibroblast cell, e.g., fibroblast activation protein (FAP).

The invention provides a method of treating an autoimmune disorder in apatient in need thereof, said method comprising administering to saidpatient a therapeutically effective amount of one or more humanizedantibodies of the invention. The invention also provides a method oftreating an autoimmune disorder in a patient in need thereof, saidmethod further comprising administering to said patient atherapeutically effective amount of one or more anti-inflammatoryagents, and/or one or more immunomodulatory agents.

The invention also provides a method of treating an inflammatorydisorder in a patient in need thereof, said method comprisingadministering to said patient a therapeutically effective amount of oneor more humanized antibodies of the invention. The invention alsoprovides a method of treating an inflammatory disorder in a patient inneed thereof, said method further comprising administering to saidpatient a therapeutically effective amount of one or moreanti-inflammatory agents, and/or one or more immunomodulatory agents.

The invention provides a method of enhancing an immune response to avaccine composition in a subject, said method comprising administeringto said subject a humanized antibody or an antigen-binding fragmentthereof that specifically binds FcγRIIB with greater affinity than saidantibody or a fragment thereof binds FcγRIIA, and a vaccine composition,such that said antibody or a fragment thereof is administered in anamount effective to enhance the immune response to said vaccinecomposition in said subject. The humanized antibodies of the inventionmay be used to enhance a humoral and/or cell mediated response againstthe antigen(s) of the vaccine composition. The antibodies of theinvention may be used in combination with any vaccines known in the art.The invention encompasses the use of the humanized antibodies of theinvention to either prevent or treat a particular disorder, where anenhanced immune response against a particular antigen or antigens iseffective to treat or prevent the disease or disorder.

The invention also provides a method for enhancing immune therapy for aninfectious agent wherein the humanized antibodies of the invention areadministered to a patient that is already infected by a pathogen, suchas HIV, HCV or HSV, to enhance opsonization and phagocytosis of infectedcells. In yet other embodiments, the invention encompasses method fortreating sepsis or septic shock using the humanized antibodies of theinvention. The role of FcγRIIB in sepsis has been described inClatworthy et al. (2004) “FcγRIIb Balances Efficient Pathogen Clearanceand the Cytokine-mediated Consequences of Sepsis,” J Exp Med199:717-723.

The invention provides a method of treating diseases with impairedapoptotic mediated signaling, e.g., cancer, autoimmune disease In aspecific embodiment, the invention encompasses a method of treating adisease with deficient Fas-mediated apoptosis, said method comprisingadministering a humanized antibody of the invention in combination withan anti-Fas antibody.

The invention further provides a method for treating or preventing anIgE-mediated allergic disorder in a patient in need thereof, comprisingadministering to said patient a therapeutically effective amount of thehumanized agonistic antibodies of the invention. The invention alsoprovides a method for treating or preventing an IgE-mediated allergicdisorder in a patient in need thereof, comprising administering to saidpatient the humanized antibodies of the invention in combination withother therapeutic antibodies or vaccine compositions used for thetreatment or prevention of IgE-mediated allergic disorders.

In another embodiment, the invention provides a method of diagnosis ofan autoimmune disease in a subject comprising:

-   -   (i) contacting a biological sample from said subject with an        effective amount of a humanized antibody of the invention; and    -   (ii) detecting binding of said humanized antibody or a fragment        thereof, wherein detection of said detectable marker above a        background or standard level indicates that said subject has an        autoimmune disease.

The invention further provides a pharmaceutical composition comprising:

-   -   (i) a therapeutically effective amount of a humanized antibody        or a fragment thereof that specifically binds FcγRIIB with        greater affinity than said antibody or a fragment thereof binds        FcγRIIA; and    -   (ii) a pharmaceutically acceptable carrier.        The invention additionally provides a pharmaceutical composition        comprising:    -   (i) a therapeutically effective amount of a humanized antibody        or a fragment thereof that specifically binds FcγRIIB with        greater affinity than said antibody or a fragment thereof binds        FcγRIIA;    -   (ii) a cytotoxic antibody that specifically binds a cancer        antigen; and    -   (iii) a pharmaceutically acceptable carrier.

In certain embodiments of the invention, pharmaceutical compositions areprovided for use in accordance with the methods of the invention, saidpharmaceutical compositions comprising a humanized FcγRIIB antibody oran antigen-binding fragment thereof, in an amount effective to prevent,treat, manage, or ameliorate a B-cell malignancy, or one or moresymptoms thereof, and a pharmaceutically acceptable carrier. Theinvention also provides pharmaceutical compositions for use inaccordance with the methods of the invention, said pharmaceuticalcompositions comprising a humanized FcγRIIB antibody or anantigen-binding fragment thereof, a prophylactic or therapeutic agentother than a FcγRIIB antagonist, and a pharmaceutically acceptablecarrier.

I. DEFINITIONS

As used herein, the term “specifically binds to FcγRIIB” and analogousterms refer to antibodies or fragments thereof (or any other FcγRIIBbinding molecules) that specifically bind to FcγRIIB or a fragmentthereof and do not specifically bind to other Fc receptors, inparticular to FcγRIIA. Further it is understood to one skilled in theart, that an antibody that specifically binds to FcγRIIB, may bindthrough the variable domain. If the antibody that specifically binds toFcγRIIB binds through its variable domain, it is understood to oneskilled in the art that it is not aggregated, i.e., is monomeric. Anantibody that specifically binds to FcγRIIB may bind to other peptidesor polypeptides with lower affinity as determined by, e.g.,immunoassays, BIAcore, or other assays known in the art. Preferably,antibodies or fragments that specifically bind to FcγRIIB or a fragmentthereof do not cross-react with other antigens. Antibodies or fragmentsthat specifically bind to FcγRIIB can be identified, for example, byimmunoassays, BIAcore, or other techniques known to those of skill inthe art. An antibody or a fragment thereof binds specifically to aFcγRIIB when it binds to FcγRIIB with higher affinity than to anycross-reactive antigen as determined using experimental techniques, suchas western blots, radioimmunoassays (RIA) and enzyme-linkedimmunosorbent assays (ELISAs). See, e.g., Paul, ed., 1989, FUNDAMENTALIMMUNOLOGY, 2^(nd) Ed., Raven Press, New York, pages 332-336.

As used herein, the term “native FcγRIIB” refers to FcγRIIB which isendogenously expressed and present on the surface of a cell. In someembodiments, “native FcγRIIB” encompasses a protein that isrecombinantly expressed in a mammalian cell. Preferably, the nativeFcγRIIB is not expressed in a bacterial cell, i.e., E. coli. Mostpreferably the native FcγRIIB is not denatured, i.e., it is in itsbiologically active conformation.

As used herein, the term “native FcγRIIA” refers to FcγRIIA which isendogenously expressed and present on the surface of a cell. In someembodiments, “native FcγRIIA” encompasses a protein that isrecombinantly expressed in a mammalian cell. Preferably, the nativeFcγRIIA is not expressed in a bacterial cell, i.e., E. coli. Mostpreferably the native FcγRIIA is not denatured, i.e., it is in itsbiologically active conformation.

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, synthetic antibodies, chimeric antibodies,camelized antibodies, single-chain Fvs (scFv), single chain antibodies,Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv),intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g.,anti-Id and anti-anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁and IgA₂) or subclass.

As used herein, the terms “B-cell malignancies” and “B-cell malignancy”refer to any B-cell lymphoproliferative disorder. B-cell malignanciesinclude tumors of B-cell origin. B-cell malignancies include, but arenot limited to, lymphomas, chronic lymphocytic leukemias, acutelymphoblastic leukemias, multiple myeloma, Hodgkin's and non-Hodgkin'sdisease, diffuse large B cell lymphoma, follicular lymphoma with areasof diffuse large B cell lymphoma, small lymphocytic lymphoma, mantlecell lymphoma, and diffuse small cleaved cell lymphoma.

As used herein, the term “derivative” refers to an antibody thatcomprises an amino acid sequence which has been altered by theintroduction of amino acid residue substitutions, deletions oradditions. The term “derivative” as used herein also refers to anantibody which has been modified, i.e., by the covalent attachment ofany type of molecule to the antibody. For example, but not by way oflimitation, an antibody may be modified, e.g., by glycosylation,acetylation, pegylation, phosphorylation, amidation, derivatization byknown protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other protein, etc. A derivative antibody may beproduced by chemical modifications using techniques known to those ofskill in the art, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Further, a derivative antibody possesses a similar or identicalfunction as the antibody from which it was derived.

The term “derivative” as used herein in conjunction with FcγRIIB refersto an antibody that immunospecifically binds to a FcγRIIB polypeptide,or an antibody fragment that immunospecifically binds to a FcγRIIBpolypeptide, that has been altered by the introduction of amino acidresidue substitutions, deletions or additions (i.e., mutations). In someembodiments, an antibody derivative or fragment thereof comprises aminoacid residue substitutions, deletions or additions in one or more CDRs.The antibody derivative may have substantially the same binding, betterbinding, or worse binding when compared to a non-derivative antibody. Inspecific embodiments, one, two, three, four, or five amino acid residuesof the CDR have been substituted, deleted or added (i.e., mutated). Theterm “derivative” as used herein in conjunction with FcγRIIB also refersto an antibody that immunospecifically binds to a FcγRIIB polypeptide,or an antibody fragment that immunospecifically binds to a FcγRIIBpolypeptide which has been modified, i.e., by the covalent attachment ofany type of molecule to the polypeptide. For example, but not by way oflimitation, an antibody, or antibody fragment may be modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Aderivative antibody, or antibody fragment may be modified by chemicalmodifications using techniques known to those of skill in the art,including, but not limited to, specific chemical cleavage, acetylation,formulation, metabolic synthesis of tunicamycin, etc. Further, aderivative of an antibody, or antibody fragment may contain one or morenon-classical amino acids. In one embodiment, an antibody derivativepossesses a similar or identical function as the parent antibody. Inanother embodiment, a derivative of an antibody, or antibody fragmenthas an altered activity when compared to an unaltered antibody. Forexample, a derivative antibody or fragment thereof can bind to itsepitope more tightly or be more resistant to proteolysis.

As used herein, the terms “disorder” and “disease” are usedinterchangeably to refer to a condition in a subject. In particular, theterm “autoimmune disease” is used interchangeably with the term“autoimmune disorder” to refer to a condition in a subject characterizedby cellular, tissue and/or organ injury caused by an immunologicreaction of the subject to its own cells, tissues and/or organs. Theterm “inflammatory disease” is used interchangeably with the term“inflammatory disorder” to refer to a condition in a subjectcharacterized by inflammation, preferably chronic inflammation.Autoimmune disorders may or may not be associated with inflammation.Moreover, inflammation may or may not be caused by an autoimmunedisorder. Thus, certain disorders may be characterized as bothautoimmune and inflammatory disorders.

As used herein, the term “cancer” refers to a neoplasm or tumorresulting from abnormal uncontrolled growth of cells. As used herein,cancer explicitly includes, leukemias and lymphomas. The term “cancer”refers to a disease involving cells that have the potential tometastasize to distal sites and exhibit phenotypic traits that differfrom those of non-cancer cells, for example, formation of colonies in athree-dimensional substrate such as soft agar or the formation oftubular networks or weblike matrices in a three-dimensional basementmembrane or extracellular matrix preparation. Non-cancer cells do notform colonies in soft agar and form distinct sphere-like structures inthree-dimensional basement membrane or extracellular matrixpreparations. Cancer cells acquire a characteristic set of functionalcapabilities during their development, albeit through variousmechanisms. Such capabilities include evading apoptosis,self-sufficiency in growth signals, insensitivity to anti-growthsignals, tissue invasion/metastasis, limitless explicative potential,and sustained angiogenesis. The term “cancer cell” is meant to encompassboth pre-malignant and malignant cancer cells. In some embodiments,cancer refers to a benign tumor, which has remained localized. In otherembodiments, cancer refers to a malignant tumor, which has invaded anddestroyed neighboring body structures and spread to distant sites. Inyet other embodiments, the cancer is associated with a specific cancerantigen.

As used herein, the term “immunomodulatory agent” and variations thereofincluding, but not limited to, immunomodulatory agents, refer to anagent that modulates a host's immune system. In certain embodiments, animmunomodulatory agent is an immunosuppressant agent. In certain otherembodiments, an immunomodulatory agent is an immunostimulatory agent.Immunomodatory agents include, but are not limited to, small molecules,peptides, polypeptides, fusion proteins, antibodies, inorganicmolecules, mimetic agents, and organic molecules.

As used herein, the term “epitope” refers to region on an antigenmolecule to which an antibody binds specifically.

As used herein, the term “fragment” refers to a peptide or polypeptidecomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least contiguous 80 amino acid residues, atleast contiguous 90 amino acid residues, at least contiguous 100 aminoacid residues, at least contiguous 125 amino acid residues, at least 150contiguous amino acid residues, at least contiguous 175 amino acidresidues, at least contiguous 200 amino acid residues, or at leastcontiguous 250 amino acid residues of the amino acid sequence of anotherpolypeptide. In a specific embodiment, a fragment of a polypeptideretains at least one function of the polypeptide. Preferably, antibodyfragments are epitope binding fragments.

As used herein, the term “humanized antibody” refers to animmunoglobulin comprising a human framework region and one or more CDR'sfrom a non-human (usually a mouse or rat) immunoglobulin. The non-humanimmunoglobulin providing the CDR's is called the “donor” and the humanimmunoglobulin providing the framework is called the “acceptor”.Constant regions need not be present, but if they are, they must besubstantially identical to human immunoglobulin constant regions, i.e.,at least about 85-90%, preferably about 95% or more identical. Hence,all parts of a humanized immunoglobulin, except possibly the CDR's, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. A “humanized antibody” is an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. For example, a humanized antibody would not encompass atypical chimeric antibody, because, e.g., the entire variable region ofa chimeric antibody is non-human. One says that the donor antibody hasbeen “humanized”, by the process of “humanization”, because theresultant humanized antibody is expected to bind to the same antigen asthe donor antibody that provides the CDR's. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichhypervariable region residues of the recipient are replaced byhypervariable region residues from a non-human species (donor antibody)such as mouse, rat, rabbit or a non-human primate having the desiredspecificity, affinity, and capacity. In some instances, Framework Region(FR) residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues which are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FRs are those of a human immunoglobulinsequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin that immunospecifically binds to a FcγRIIBpolypeptide, that has been altered by the introduction of amino acidresidue substitutions, deletions or additions (i.e., mutations). In someembodiments, a humanized antibody is a derivative. Such a humanizedantibody comprises amino acid residue substitutions, deletions oradditions in one or more non-human CDRs. The humanized antibodyderivative may have substantially the same binding, better binding, orworse binding when compared to a non-derivative humanized antibody. Inspecific embodiments, one, two, three, four, or five amino acid residuesof the CDR have been substituted, deleted or added (i.e., mutated). Forfurther details in humanizing antibodies, see European Patent Nos. EP239,400, EP 592,106, and EP 519,596; International Publication Nos. WO91/09967 and WO 93/17105; U.S. Pat. Nos. 5,225,539, 5,530,101,5,565,332, 5,585,089, 5,766,886, and 6,407,213; and Padlan (1991) “APossible Procedure For Reducing The Immunogenicity Of Antibody VariableDomains While Preserving Their Ligand-Binding Properties,” MolecularImmunology 28:489-498; Studnicka et al. (1994) “Human-EngineeredMonoclonal Antibodies Retain Full Specific Binding Activity ByPreserving Non-CDR Complementarity-Modulating Residues,” ProteinEngineering 7:805-814; Roguska et al. (1994) “Humanization Of MurineMonoclonal Antibodies Through Variable Domain Resurfacing,” Proc. Nat.Acad. Sci. 91:969-973; Tan et al. (2002) “‘Superhumanized’ Antibodies:Reduction Of Immunogenic Potential By Complementarity-Determining RegionGrafting With Human Germline Sequences: Application To An Anti-CD28,” J.Immunol. 169:1119-1125; Caldas et al. (2000) “Design And Synthesis OfGermline-Based Hemi-Humanized Single-Chain Fv Against The CD18 SurfaceAntigen,” Protein Eng. 13:353-360; Morea et al. (2000) “AntibodyModeling: Implications For Engineering And Design,” Methods 20:267-279;Baca et al. (1997) “Antibody Humanization Using Monovalent PhageDisplay,” J. Biol. Chem. 272:10678-10684; Roguska et al. (1996) “AComparison Of Two Murine Monoclonal Antibodies Humanized By CDR-GraftingAnd Variable Domain Resurfacing,” Protein Eng. 9:895-904; Couto et al.(1995) “Designing Human Consensus Antibodies With Minimal PositionalTemplates,” Cancer Res. 55:5973s-5977s; Couto et al. (1995) “Anti-BA46Monoclonal Antibody Mc3: Humanization Using A Novel Positional ConsensusAnd In Vivo And In Vitro Characterization,” Cancer Res. 55:1717-1722;Sandhu (1994) “A Rapid Procedure For The Humanization Of MonoclonalAntibodies,” Gene 150:409-410; Pedersen et al. (1994) “Comparison ofSurface Accessible Residues in Human and Murine Immunoglobulin FvDomains: Implication for Humanization of Murine Antibodies,” J. Mol.Biol. 235:959-973; Jones et al. (1986) “Replacing TheComplementarity-Determining Regions In A Human Antibody With Those FromA Mouse,” Nature 321:522-525; Reichmann et al, (1988) “Reshaping HumanAntibodies For Therapy,” Nature 332:323-329; and Presta (1992) “Antibodyengineering,” Curr. Op. Biotechnol. 3:394-8.

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody which are responsible for antigen binding. Thehypervariable region comprises amino acid residues from a“Complementarity Determining Region” or “CDR” (i.e., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST,5^(th) Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) and/or those residues from a “hypervariable loop”(i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavychain variable domain; Chothia and Lesk, 1987, J. Mol. Biol.196:901-917). CDR residues for Eph099B-208.261 and Eph099B-233.152 arelisted in Table 1. “Framework Region” or “FR” residues are thosevariable domain residues other than the hypervariable region residues asherein defined.

As used herein, the terms “single-chain Fv” or “scFv” refer to antibodyfragments comprise the VH and VL domains of antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. For a review of sFv, see Pluckthun in THE PHARMACOLOGY OFMONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore, eds.Springer-Verlag, New York, pp. 269-315 (1994). In specific embodiments,scFvs include bi-specific scFvs and humanized scFvs.

As used herein, the terms “nucleic acids” and “nucleotide sequences”include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g.,mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNAmolecules, and analogs of DNA or RNA molecules. Such analogs can begenerated using, for example, nucleotide analogs, which include, but arenot limited to, inosine or tritylated bases. Such analogs can alsocomprise DNA or RNA molecules comprising modified backbones that lendbeneficial attributes to the molecules such as, for example, nucleaseresistance or an increased ability to cross cellular membranes. Thenucleic acids or nucleotide sequences can be single-stranded,double-stranded, may contain both single-stranded and double-strandedportions, and may contain triple-stranded portions, but preferably isdouble-stranded DNA.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, a subject is preferably a mammal suchas a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and aprimate (e.g., monkey and human), most preferably a human.

As used herein, the terms “treat,” “treating” and “treatment” refer tothe eradication, reduction or amelioration of symptoms of a disease ordisorder related to the loss of regulation in the Fc receptor signalingpathway or to enhance the therapeutic efficacy of another therapy, e.g.,a therapeutic antibody, vaccine therapy or prophylaxis. In someembodiments, treatment refers to the eradication, removal, modification,or control of primary, regional, or metastatic cancer tissue thatresults from the administration of one or more therapeutic agents. Incertain embodiments, such terms refer to the minimizing or delaying thespread of cancer resulting from the administration of one or moretherapeutic agents to a subject with such a disease. In otherembodiments, such terms refer to elimination of disease causing cells.

As used herein, the phrase “side effects” encompasses unwanted andadverse effects of a prophylactic or therapeutic agent. Adverse effectsare always unwanted, but unwanted effects are not necessarily adverse.An adverse effect from a prophylactic or therapeutic agent might beharmful or uncomfortable or risky. Side effects from chemotherapyinclude, but are not limited to, gastrointestinal toxicity such as, butnot limited to, early and late-forming diarrhea and flatulence, nausea,vomiting, anorexia, leukopenia, anemia, neutropenia, asthenia, abdominalcramping, fever, pain, loss of body weight, dehydration, alopecia,dyspnea, insomnia, dizziness, mucositis, xerostomia, and kidney failure,as well as constipation, nerve and muscle effects, temporary orpermanent damage to kidneys and bladder, flu-like symptoms, fluidretention, and temporary or permanent infertility. Side effects fromradiation therapy include but are not limited to fatigue, dry mouth, andloss of appetite. Side effects from biological therapies/immunotherapiesinclude but are not limited to rashes or swellings at the site ofadministration, flu-like symptoms such as fever, chills and fatigue,digestive tract problems and allergic reactions. Side effects fromhormonal therapies include but are not limited to nausea, fertilityproblems, depression, loss of appetite, eye problems, headache, andweight fluctuation. Additional undesired effects typically experiencedby patients are numerous and known in the art, see, e.g., thePHYSICIANS' DESK REFERENCE (56^(th) ed., 2002), which is incorporatedherein by reference in its entirety.

As used herein, a “therapeutically effective amount” refers to thatamount of the therapeutic agent sufficient to treat or manage a diseaseor disorder associated with FcγRIIB and any disease related to the lossof regulation in the Fc receptor signaling pathway or to enhance thetherapeutic efficacy of another therapy, e.g., therapeutic antibody,vaccine therapy or prophylaxis, etc. A therapeutically effective amountmay refer to the amount of therapeutic agent sufficient to delay orminimize the onset of disease, e.g., delay or minimize the spread ofcancer. A therapeutically effective amount may also refer to the amountof the therapeutic agent that provides a therapeutic benefit in thetreatment or management of a disease. Further, a therapeuticallyeffective amount with respect to a therapeutic agent of the inventionmeans that amount of therapeutic agent alone, or in combination withother therapies, that provides a therapeutic benefit in the treatment ormanagement of a disease, e.g., sufficient to enhance the therapeuticefficacy of a therapeutic antibody sufficient to treat or manage adisease. Used in connection with an amount of FcγRIIB antibody of theinvention, the term can encompass an amount that improves overalltherapy, reduces or avoids unwanted effects, or enhances the therapeuticefficacy of or synergies with another therapeutic agent.

As used herein, the terms “prophylactic agent” and “prophylactic agents”refer to any agent(s) which can be used in the prevention of a disorder,or prevention of recurrence or spread of a disorder. A prophylacticallyeffective amount may refer to the amount of prophylactic agentsufficient to prevent the recurrence or spread of hyperproliferativedisease, particularly cancer, or the occurrence of such in a patient,including but not limited to those predisposed to hyperproliferativedisease, for example those genetically predisposed to cancer orpreviously exposed to carcinogens. A prophylactically effective amountmay also refer to the amount of the prophylactic agent that provides aprophylactic benefit in the prevention of disease. Further, aprophylactically effective amount with respect to a prophylactic agentof the invention means that amount of prophylactic agent alone, or incombination with other agents, that provides a prophylactic benefit inthe prevention of disease. Used in connection with an amount of anFcγRIIB antibody of the invention, the term can encompass an amount thatimproves overall prophylaxis or enhances the prophylactic efficacy of orsynergies with another prophylactic agent, such as but not limited to atherapeutic antibody. In certain embodiments, the term “prophylacticagent” refers to an agonistic FcγRIIB-specific antibody. In otherembodiments, the term “prophylactic agent” refers to an antagonisticFcγRIIB-specific antibody. In certain other embodiments, the term“prophylactic agent” refers to cancer chemotherapeutics, radiationtherapy, hormonal therapy, biological therapy (e.g., immunotherapy),and/or FcγRIIB antibodies of the invention. In other embodiments, morethan one prophylactic agent may be administered in combination.

As used herein, the terms “manage,” “managing” and “management” refer tothe beneficial effects that a subject derives from administration of aprophylactic or therapeutic agent, which does not result in a cure ofthe disease. In certain embodiments, a subject is administered one ormore prophylactic or therapeutic agents to “manage” a disease so as toprevent the progression or worsening of the disease.

As used herein, the terms “prevent”, “preventing” and “prevention” referto the prevention of the occurrence and/or recurrence or onset of one ormore symptoms of a disorder in a subject resulting from theadministration of a prophylactic or therapeutic agent.

As used herein, the term “in combination” refers to the use of more thanone prophylactic and/or therapeutic agents. The use of the term “incombination” does not restrict the order in which prophylactic and/ortherapeutic agents are administered to a subject with a disorder, e.g.,hyperproliferative cell disorder, especially cancer. A firstprophylactic or therapeutic agent can be administered prior to (e.g., 1minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours,4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeksbefore), concomitantly with, or subsequent to (e.g., 1 minute, 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after)the administration of a second prophylactic or therapeutic agent to asubject which had, has, or is susceptible to a disorder. Theprophylactic or therapeutic agents are administered to a subject in asequence and within a time interval such that the agent of the inventioncan act together with the other agent to provide an increased benefitthan if they were administered otherwise. Any additional prophylactic ortherapeutic agent can be administered in any order with the otheradditional prophylactic or therapeutic agents.

II. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B: Amino Acid Alignments.

FIG. 1A. The alignment of the amino acid sequences of mouse 2B6 VH (SEQID NO:28), humanized 2B6 VH1 (SEQ ID NO:24), and human VH1-18 and humanJH6 (SEQ ID NO:60): is shown in FIG. 1A.

FIG. 1B. This figure shows the alignment of amino acid sequences ofmurine 2B6VL (SEQ ID NO:26), human 2B6VL-1 (SEQ ID NO:18), human 2B6VL-2(SEQ ID NO:20); human 2B6VL-3 (SEQ ID NO:22), and human VKA26 and humanJκ4 (SEQ ID NO:61).

FIG. 2: Binding Of Hu2b6hc/Ch2b6lc Mab And Ch2b6 Mab To FcγRiib.

Binding to dimeric soluble FcγRIIB-Fc was determined by ELISA.hu2B6HC/ch2B6LC monoclonal antibody bound to the receptor with similaraffinity to the ch2B6 monoclonal antibody.

FIG. 3: Binding OF hu2B6LC/ch2B6HC mAB, ch2B6LC/hu2B6HC, AND ch2B6 mAbTO FcγRIIB.

Binding to dimeric soluble FcγRIIB-Fc was determined by ELISA.hu2B6HC/ch2B6LC mAb and ch2B6HC/hu2B6LC mAB bound to the receptor withsimilar affinity to the ch2B6 mAb.

FIG. 4: Binding OF hu2B6 Variants To FcγRIIB.

Binding of Hu2B6N50Y; Hu2B6N50Y,V51A; Ch2B6, and Hu2B6 to dimericsoluble FcγRIIb-Fc was determined by ELISA. All of the mAbs bound to thereceptor with similar affinity.

FIG. 5: Binding Of hu2B6 Variants To FcγRIIA.

Binding of Hu2B6N50Y; Hu2B6N50Y,V51A; Ch2B6, and Hu2B6 to dimericsoluble FcγRIIa-Fc was determined by ELISA. The humanized 2B6 mAbsselectively bind to CD32B. All of the solid data points fall on top ofeach other and are only displayed as a solid square.

DESCRIPTION OF THE PREFERRED EMBODIMENTS I. FcγRIIB-Specific Antibodies

The present invention encompasses humanized antibodies (preferablyhumanized monoclonal antibodies) or fragments thereof that specificallybind FcγRIIB, preferably human FcγRIIB, more preferably native humanFcγRIIB with a greater affinity than said antibodies or fragmentsthereof bind FcγRIIA, preferably human FcγRIIA, more preferably nativehuman FcγRIIA. Preferably, the humanized antibodies of the inventionbind the extracellular domain of native human FcγRIIB. In certainembodiments, the humanized antibodies or fragments thereof bind toFcγRIIB with an affinity greater than two-fold, four fold, 6 fold, 10fold, 20 fold, 50 fold, 100 fold, 1000 fold, 10⁴ fold, 10⁵ fold, 10⁶fold, 10⁷ fold, or 10⁸ fold than said antibodies or fragments thereofbind FcγRIIA. In one particular embodiment, the humanized antibody ofthe invention is derived from a mouse monoclonal antibody produced byclone 2B6 or 3H7, having ATCC accession numbers PTA-4591 and PTA-4592,respectively. In another embodiment, the humanized antibody of theinvention is derived from a mouse monoclonal antibody produced by clone1D5, 2E1, 2H9, 2D11, or 1F2, having ATCC Accession numbers, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively. Hybridomasproducing antibodies 2B6 and 3H7 have been deposited with the AmericanType Culture Collection (10801 University Blvd., Manassas, Va.20110-2209) on Aug. 13, 2002 under the provisions of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedures, and assigned accession numbersPTA-4591 (for hybridoma producing 2B6) and PTA-4592 (for hybridomaproducing 3H7), respectively, and are incorporated herein by reference.Hybridomas producing 1D5, 2E1, 2H9, 2D11, and 1F2 were deposited underthe provisions of the Budapest Treaty with the American Type CultureCollection (10801 University Blvd., Manassas, Va. 20110-2209) on May 7,2004, and assigned accession numbers PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively and are incorporated herein byreference.

In yet other embodiments, the invention encompasses humanized FcγRIIBantibodies that bind exclusively to FcγRIIB and have no affinity forFcγRIIA using standard methods known in the art and disclosed herein.

In a specific embodiment, the invention encompasses a humanized antibodycomprising the CDRs of 2B6 or of 3H7. In particular, an antibody withthe heavy chain variable domain having the amino acid sequence of SEQ IDNO:24 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In a specificembodiment, the invention encompasses a humanized antibody with theheavy chain variable domain having the amino acid sequence of SEQ IDNO:37 and the light chain variable domain having the amino acid sequenceof SEQ ID NO:46. In yet another preferred embodiment, the humanizedantibodies of the invention further do not bind Fc activation receptors,e.g., FcγIIIA, FcγIIIB, etc. In one embodiment, the humanizedFcγRIIB-specific antibody in accordance with the invention is notderived from the monoclonal antibody designated KB61, as disclosed inPulford et al. (1986) “A New Monoclonal Antibody (KB61) Recognizing ANovel Antigen Which Is Selectively Expressed On A Subpopulation Of HumanB Lymphocytes,” Immunology, 57:71-76 or the monoclonal antibodydesignated MAbII8D2 as disclosed in Weinrich et al. (1996) “EpitopeMapping Of New Monoclonal Antibodies Recognizing Distinct Human FcRII(CD32) Isoforms,” Hybridoma 15: 109-116. In a specific embodiment, theFcγRIIB-specific antibody of the invention does not bind to the sameepitope and/or does not compete with binding with the monoclonalantibody KB61 or II8D2. Preferably, the humanized FcγRIIB-specificantibodies of the invention do not bind the amino acid sequence SDPNFSI(SEQ ID NO:59) corresponding to positions 135-141 of FcγRIIB2 isoform.

The constant domains of the humanized antibodies of the invention may beselected with respect to the proposed function of the antibody, inparticular with regard to the effector function which may be required.In some embodiments, the constant domains of the humanized antibodies ofthe invention are human IgA, IgE, IgG or IgM domains. In a specificembodiment, human IgG constant domains, especially of the IgG1 and IgG3isotypes are used, especially when the humanized antibodies of theinvention are intended for therapeutic uses and antibody effectorfunctions are needed. In alternative embodiments, IgG2 and IgG4 isotypesare used when the humanized antibody of the invention is intended fortherapeutic purposes and antibody effector function is not required. Inother embodiments, the invention encompasses humanized antibodiescomprising one or more amino acid modifications in the Fc region such asthose disclosed in U.S. Patent Application Publication Nos. 2005/0037000and 2005/0064514, by Stavenhagen et al.; U.S. Provisional ApplicationNos. 60/439,498; 60/456,041; and 60/514,549 filed on Jan. 9, 2003; Mar.19, 2003, and Oct. 23, 2003 respectively; and U.S. Pat. Nos. 5,624,821and 5,648,260 and European Patent No. EP 0 307 434; all of which areincorporated herein by reference in their entireties.

Preferably the humanized antibodies of the invention bind theextracellular domain of native human FcγRIIB. The humanized anti-FcγRIIBantibodies of the invention may have a heavy chain variable regioncomprising the amino acid sequence of CDR1 (SEQ ID NO:1 or SEQ ID NO:29)and/or CDR2 (SEQ ID NO:2 or SEQ ID NO:30) and/or CDR3 (SEQ ID NO:3 orSEQ ID NO:31) and/or a light chain variable region comprising the aminoacid sequence of CDR1 (SEQ ID NO:8 or SEQ ID NO:38) and/or a CDR2 (SEQID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:39) and/or CDR3 (SEQID NO:12 or SEQ ID NO:40).

In one specific embodiment, the invention provides a humanized 2B6antibody, wherein the VH region consists of the FR segments from thehuman germline VH segment VH1-18 (Matsuda et al. (1998) “The CompleteNucleotide Sequence of the Human Immunoglobulin Heavy Chain VariableRegion Locus,” J. Exp. Med. 188:2151-2162) and JH6 (Ravetch et al.(1981) “Structure Of The Human Immunoglobulin Mu Locus: CharacterizationOf Embryonic And Rearranged J And D Genes,” Cell 27:583-591), and one ormore CDR regions of the 2B6 VH, having the amino acid sequence of SED IDNO:1, SEQ ID NO:2, or SEQ ID NO:3. In one embodiment, the 2B6 VH has theamino acid sequence of SEQ ID NO:24. In another specific embodiment, thehumanized 2B6 antibody further comprises a VL region, which consists ofthe FR segments of the human germline VL segment VK-A26 (Lautner-Rieskeet al. (1992) “The Human Immunoglobulin Kappa Locus. Characterization ofthe Duplicated A Regions,” Eur. J. Immunol. 22:1023-1029) and JK4(Hieter et al. (1982) “Evolution Of Human Immunoglobulin Kappa J RegionGenes,” J. Biol. Chem. 257:1516-1522), and one or more CDR regions of2B6VL, having the amino acid sequence of SEQ ID NO:8, SEQ ID NO:9, SEQID NO:10, SEQ ID NO:11, and SEQ ID NO:12. In one embodiment, the 2B6 VLhas the amino acid sequence of SEQ ID NO:18; SEQ ID NO:20, or SEQ IDNO:22. The antibodies used in the methods of the invention may be fromany animal origin including birds and mammals (e.g., human, non-humanprimate, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse,or chicken). Preferably, the antibodies are human or humanizedmonoclonal antibodies. As used herein, “human” antibodies includeantibodies having the amino acid sequence of a human immunoglobulin andinclude antibodies isolated from human immunoglobulin libraries orlibraries of synthetic human immunoglobulin coding sequences or frommice that express antibodies from human genes.

In another specific embodiment, the invention provides a humanized 3H7antibody, wherein the VH region consists of the FR segments from a humangermline VH segment and the CDR regions of the 3H7 VH, having the aminoacid sequence of SEQ ID NO:37. In another specific embodiment, thehumanized 3H7 antibody further comprises a VL regions, which consists ofthe FR segments of a human germline VL segment and the CDR regions of3H7VL, having the amino acid sequence of SEQ ID NO:46.

In particular, the invention provides a humanized antibody thatimmunospecifically binds to extracellular domain of native humanFcγRIIB, said antibody comprising (or alternatively, consisting of) CDRsequences of 2B6 or 3H7, in any of the following combinations: a VH CDR1and a VL CDR1; a VH CDR1 and a VL CDR2; a VH CDR1 and a VL CDR3; a VHCDR2 and a VL CDR1; VH CDR2 and VL CDR2; a VH CDR2 and a VL CDR3; a VHCDR3 and a VH CDR1; a VH CDR3 and a VL CDR2; a VH CDR3 and a VL CDR3; aVH1 CDR1, a VH CDR2 and a VL CDR1; a VH CDR1, a VH CDR2 and a VL CDR2; aVH CDR1, a VH CDR2 and a VL CDR3; a VH CDR2, a VH CDR3 and a VL CDR1, aVH CDR2, a VH CDR3 and a VL CDR2; a VH CDR2, a VH CDR2 and a VL CDR3; aVH CDR1, a VL CDR1 and a VL CDR2; a VH CDR1, a VL CDR1 and a VL CDR3; aVH CDR2, a VL CDR1 and a VL CDR2; a VH CDR2, a VL CDR1 and a VL CDR3; aVH CDR3, a VL CDR1 and a VL CDR2; a VH CDR3, a VL CDR1 and a VL CDR3; aVH CDR1, a VH CDR2, a VH CDR3 and a VL CDR1; a VH CDR1, a VH CDR2, a VHCDR3 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3 and a VL CDR3; a VHCDR1, a VH CDR2, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR3, a VL CDR1 and a VL CDR2; a VHCDR1, a VH CDR3, a VL CDR1 and a VL CDR3; a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR2; a VH CDR2, a VH CDR3, a VL CDR1 and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR2 and a VL CDR3; a VH CDR1, a VH CDR2, a VHCDR3, a VL CDR1 and a VL CDR2; a VH CDR1, a VH CDR2, a VH CDR3, a VLCDR1 and a VL CDR3; a VH CDR1, a VH CDR2, a VL CDR1, a VL CDR2, and a VLCDR3; a VH CDR1, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; a VHCDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3; or any combinationthereof of the VH CDRs and VL CDRs disclosed herein.

The present invention provides humanized antibody molecules specific forFcγRIIB in which one or more regions of one or more CDRs of the heavyand/or light chain variable regions of a human antibody (the recipientantibody) have been substituted by analogous parts of one or more CDRsof a donor monoclonal antibody which specifically binds FcγRIIB, with agreater affinity than FcγRIIA, e.g., a monoclonal antibody produced byclone 2B6 or 3H7, having ATCC accession numbers PTA-4591, and PTA-4592,respectively, or a monoclonal antibody produced by clone 1D5, 2E1, 2H9,2D11, or 1F2, having ATCC Accession numbers, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively. In other embodiments,the humanized antibodies of the invention bind to the same epitope as2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. In a most preferred embodiment,the humanized antibody specifically binds to the same epitope as thedonor murine antibody. It will be appreciated by one skilled in the artthat the invention encompasses CDR grafting of antibodies in general.Thus, the donor and acceptor antibodies may be derived from animals ofthe same species and even same antibody class or sub-class. Moreusually, however, the donor and acceptor antibodies are derived fromanimals of different species. Typically the donor antibody is anon-human antibody, such as a rodent MAb, and the acceptor antibody is ahuman antibody.

In some embodiments, at least one CDR from the donor antibody is graftedonto the human antibody. In other embodiments, at least two andpreferably all three CDRs of each of the heavy and/or light chainvariable regions are grafted onto the human antibody. The CDRs maycomprise the Kabat CDRs, the structural loop CDRs or a combinationthereof. In some embodiments, the invention encompasses a humanizedFcγRIIB antibody comprising at least one CDR grafted heavy chain and atleast one CDR-grafted light chain.

In a preferred embodiment, the CDR regions of the humanized FcγRIIBspecific antibody are derived from a murine antibody specific forFcγRIIB. In some embodiments, the humanized antibodies described hereincomprise alterations, including but not limited to amino acid deletions,insertions, modifications, of the acceptor antibody, i.e., human, heavyand/or light chain variable domain framework regions that are necessaryfor retaining binding specificity of the donor monoclonal antibody. Insome embodiments, the framework regions of the humanized antibodiesdescribed herein does not necessarily consist of the precise amino acidsequence of the framework region of a natural occurring human antibodyvariable region, but contains various alterations, including but notlimited to amino acid deletions, insertions, modifications that alterthe property of the humanized antibody, for example, improve the bindingproperties of a humanized antibody region that is specific for the sametarget as the murine FcγRIIB specific antibody. In most preferredembodiments, a minimal number of alterations are made to the frameworkregion in order to avoid large-scale introductions of non-humanframework residues and to ensure minimal immunogenicity of the humanizedantibody in humans. The donor monoclonal antibody of the presentinvention is preferably a monoclonal antibody produced by clones 2B6 and3H7 (having ATCC accession numbers PTA-4591, and PTA-4592, respectively)which bind FcγRIIB or a monoclonal antibody produced by clones 1D5, 2E1,2H9, 2D11, or 1F2 (having ATCC Accession numbers, PTA-5958, PTA-5961,PTA-5962, PTA-5960, and PTA-5959, respectively).

In a specific embodiment, the invention encompasses a CDR-graftedantibody which specifically binds FcγRIIB with a greater affinity thansaid antibody binds FcγRIIA, wherein the CDR-grafted antibody comprisesa heavy chain variable region domain comprising framework residues ofthe recipient antibody and residues from the donor monoclonal antibody,which specifically binds FcγRIIB with a greater affinity than saidantibody binds FcγRIIA, e.g., monoclonal antibody produced from clones2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2. In another specific embodiment,the invention encompasses a CDR-grafted antibody which specificallybinds FcγRIIB with a greater affinity than said antibody binds FcγRIIA,wherein the CDR-grafted antibody comprises a light chain variable regiondomain comprising framework residues of the recipient antibody andresidues from the donor monoclonal antibody, which specifically bindsFcγRIIB with a greater affinity than said antibody binds FcγRIIA, e.g.,monoclonal antibody produced from clones 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2.

A humanized FcγRIIB specific antibody of the invention may comprisesubstantially all of at least one, and typically two, variable domainsin which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin (i.e., donor antibody) and all orsubstantially all of the framework regions are those of a humanimmunoglobulin consensus sequence. Preferably, a humanized antibody ofthe invention also comprises at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Theconstant domains of the humanized antibodies of the invention may beselected with respect to the proposed function of the antibody, inparticular the effector function which may be required. In someembodiments, the constant domains of the humanized antibodies of theinvention are human IgA, IgE, IgG or IgM domains. In a specificembodiment, human IgG constant domains, especially of the IgG1 and IgG3isotypes are used, when the humanized antibodies of the invention isintended for therapeutic uses and antibody effector functions areneeded. In alternative embodiments, IgG2 and IgG4 isotypes are used whenthe humanized antibody of the invention is intended for therapeuticpurposes and antibody effector function is not required. The inventionencompasses Fc constant domains comprising one or more amino acidmodifications which alter antibody effector functions such as thosedisclosed in U.S. Patent Application Publication Nos. 2005/0037000 and2005/0064514; U.S. Provisional Application Nos. 60/439,498; 60/456,041;and 60/514,549 filed on Jan. 9, 2003; Mar. 19, 2003, and Oct. 23, 2003respectively; all of which are incorporated herein by reference in theirentireties.

In some embodiments, the humanized antibody of the invention containsboth the light chain as well as at least the variable domain of a heavychain. In other embodiments, the humanized antibody of the invention mayfurther comprise one or more of the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. The humanized antibody can be selected fromany class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, andany isotype, including IgG₁, IgG₂, IgG₃ and IgG₄. In some embodiments,the constant domain is a complement fixing constant domain where it isdesired that the humanized antibody exhibit cytotoxic activity, and theclass is typically IgG₁. In other embodiments, where such cytotoxicactivity is not desirable, the constant domain may be of the IgG₂ class.The humanized antibody of the invention may comprise sequences from morethan one class or isotype, and selecting particular constant domains tooptimize desired effector functions is within the ordinary skill in theart.

The framework and CDR regions of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor CDR orthe consensus framework may be mutagenized by substitution, insertion ordeletion of at least one residue so that the CDR or framework residue atthat site does not correspond to either the consensus or the donorantibody. Such mutations, however, are preferably not extensive.Usually, at least 75% of the humanized antibody residues will correspondto those of the parental framework region (FR) and CDR sequences, moreoften 90%, and most preferably greater than 95%. Humanized antibodiescan be produced using variety of techniques known in the art, including,but not limited to, CDR-grafting (European Patent No. EP 239,400;International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539,5,530,101, and 5,585,089), veneering or resurfacing (European PatentNos. EP 592,106 and EP 519,596; Padlan (1991) “A Possible Procedure ForReducing The Immunogenicity Of Antibody Variable Domains WhilePreserving Their Ligand-Binding Properties,” Molecular Immunology28(4/5):489-498; Studnicka et al. (1994) “Human-Engineered MonoclonalAntibodies Retain Full Specific Binding Activity By Preserving Non-CdrComplementarity-Modulating Residues,” Protein Engineering 7:805-814; andRoguska et al. (1994) “Humanization Of Murine Monoclonal AntibodiesThrough Variable Domain Resurfacing,” Proc. Nat. Acad. Sci. 91:969-973),chain shuffling (U.S. Pat. No. 5,565,332), and techniques disclosed in,e.g., U.S. Pat. Nos. 6,407,213, 5,766,886, 5,585,089, InternationalPublication No. WO 9317105, Tan et al. (2002) “‘Superhumanized’Antibodies: Reduction Of Immunogenic Potential ByComplementarity-Determining Region Grafting With Human GermlineSequences: Application To An Anti-CD28,” J. Immunol. 169:1119 1125;Caldas et al. (2000) “Design And Synthesis Of Germline-BasedHemi-Humanized Single-Chain Fv Against The CD18 Surface Antigen,”Protein Eng. 13:353 360; Morea et al. (2000) “Antibody Modeling:Implications For Engineering And Design,” Methods 20:267 279; Baca etal. (1997) “Antibody Humanization Using Monovalent Phage Display,” J.Biol. Chem. 272:10678-10684; Roguska et al. (1996) “A Comparison Of TwoMurine Monoclonal Antibodies Humanized By CDR-Grafting And VariableDomain Resurfacing,” Protein Eng. 9:895 904; Couto et al. (1995)“Designing Human Consensus Antibodies With Minimal PositionalTemplates,” Cancer Res. 55 (23 Supp):5973s 5977s; Couto et al. (1995)“Anti-BA46 Monoclonal Antibody Mc3: Humanization Using A NovelPositional Consensus And In Vivo And In Vitro Characterization,” CancerRes. 55:1717 22; Sandhu (1994) “A Rapid Procedure For The HumanizationOf Monoclonal Antibodies,” Gene 150:409 410; Pedersen et al. (1994)“Comparison Of Surface Accessible Residues In Human And MurineImmunoglobulin Fv Domains. Implication For Humanization Of MurineAntibodies,” J. Mol. Biol. 235:959 973; Jones et al. (1986) “ReplacingThe Complementarity-Determining Regions In A Human Antibody With ThoseFrom A Mouse,” Nature 321:522-525; Riechmann et al. (1988) “ReshapingHuman Antibodies For Therapy,” Nature 332:323-327; and Presta (1992)“Antibody Engineering,” Curr. Op. Biotech. 3:394-398. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; U.S. Publication Nos. 2004/0049014 and2003/0229208; U.S. Pat. Nos. 6,350,861; 6,180,370; 5,693,762; 5,693,761;5,585,089; and 5,530,101 and Riechmann et al., 1988, Nature 332:323, allof which are incorporated herein by reference in their entireties.)

In a particular embodiment, the humanized antibodies of the invention,or fragments thereof, agonize at least one activity of FcγRIIB. In oneembodiment of the invention, said activity is inhibition of B cellreceptor-mediated signaling. In another embodiment, the humanizedagonistic antibodies of the invention inhibit activation of B cells, Bcell proliferation, antibody production, intracellular calcium influx ofB cells, cell cycle progression, or activity of one or more downstreamsignaling molecules in the FcγRIIB signal transduction pathway. In yetanother embodiment, the humanized agonistic antibodies of the inventionenhance phosphorylation of FcγRIIB or SHIP recruitment. In a furtherembodiment of the invention, the humanized agonistic antibodies inhibitMAP kinase activity or Akt recruitment in the B cell receptor-mediatedsignaling pathway. In another embodiment, the humanized agonisticantibodies of the invention agonize FcγRIIB-mediated inhibition of FcεRIsignaling. In a particular embodiment, said humanized antibodies inhibitFcεRI-induced mast cell activation, calcium mobilization, degranulation,cytokine production, or serotonin release. In another embodiment, thehumanized agonistic antibodies of the invention stimulatephosphorylation of FcγRIIB, stimulate recruitment of SHIP, stimulateSHIP phosphorylation and its association with Shc, or inhibit activationof MAP kinase family members (e.g., Erk1, Erk2, JNK, p38, etc.). In yetanother embodiment, the humanized agonistic antibodies of the inventionenhance tyrosine phosphorylation of p62dok and its association with SHIPand rasGAP. In another embodiment, the humanized agonistic antibodies ofthe invention inhibit FcγR-mediated phagocytosis in monocytes ormacrophages.

In another embodiment, the humanized antibodies of the invention, orfragments thereof, antagonize at least one activity of FcγRIIB. In oneembodiment, said activity is activation of B cell receptor-mediatedsignaling. In a particular embodiment, the humanized antagonisticantibodies of the invention enhance B cell activity, B cellproliferation, antibody production, intracellular calcium influx, oractivity of one or more downstream signaling molecules in the FcγRIIBsignal transduction pathway. In yet another particular embodiment, thehumanized antagonistic antibodies of the invention decreasephosphorylation of FcγRIIB or SHIP recruitment. In a further embodimentof the invention, the humanized antagonistic antibodies enhance MAPkinase activity or Akt recruitment in the B cell receptor mediatedsignaling pathway. In another embodiment, the humanized antagonisticantibodies of the invention antagonize FcγRIIB-mediated inhibition ofFcεRI signaling. In a particular embodiment, the humanized antagonisticantibodies of the invention enhance FcεRI-induced mast cell activation,calcium mobilization, degranulation, cytokine production, or serotoninrelease. In another embodiment, the humanized antagonistic antibodies ofthe invention inhibit phosphorylation of FcγRIIB, inhibit recruitment ofSHIP, inhibit SHIP phosphorylation and its association with Shc, enhanceactivation of MAP kinase family members (e.g., Erk1, Erk2, JNK, p38,etc.). In yet another embodiment, the humanized antagonistic antibodiesof the invention inhibit tyrosine phosphorylation of p62dok and itsassociation with SHIP and rasGAP. In another embodiment, the humanizedantagonistic antibodies of the invention enhance FcγR-mediatedphagocytosis in monocytes or macrophages. In another embodiment, thehumanized antagonistic antibodies of the invention prevent phagocytosis,clearance of opsonized particles by splenic macrophages.

Antibodies of the invention include, but are not limited to, monoclonalantibodies, synthetic antibodies, recombinantly produced antibodies,multispecific antibodies, human antibodies, chimeric antibodies,camelized antibodies, single-chain Fvs (scFv), single chain antibodies,Fab fragments, F(ab′) fragments, disulfide-linked Fvs (sdFv),intrabodies, and epitope-binding fragments of any of the above. Inparticular, antibodies used in the methods of the present inventioninclude immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site that immunospecifically binds to FcγRIIB with greateraffinity than said immunoglobulin molecule binds FcγRIIA. Antibodyanalogs may also include FcγRIIB-specific T-cell receptors, for example,chimeric T-cell receptors (see, e.g., U.S. Patent ApplicationPublication No. 2004/0043401), a single-chain T-cell receptor linked toa single-chain antibody (see, e.g., U.S. Pat. No. 6,534,633), andprotein scaffolds (see, e.g., U.S. Pat. No. 6,818,418). In certainembodiments, an antibody analog of the invention is not a monoclonalantibody.

The humanized antibodies used in the methods of the invention may befrom any animal origin including birds and mammals (e.g., human,non-human primate, murine, donkey, sheep, rabbit, goat, guinea pig,camel, horse, or chicken). Preferably, the antibodies are human orhumanized monoclonal antibodies. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or libraries of synthetic human immunoglobulin codingsequences or from mice that express antibodies from human genes.

The humanized antibodies used in the methods of the present inventionmay be monospecific, bispecific, trispecific or of greatermultispecificity. Multispecific antibodies may immunospecifically bindto different epitopes of FcγRIIB or immunospecifically bind to both anepitope of FcγRIIB as well a heterologous epitope, such as aheterologous polypeptide or solid support material. (See, e.g.,International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360,and WO 92/05793; Tutt et al. (1991) “Trispecific F(ab′)3 DerivativesThat Use Cooperative Signaling Via The TCR/CD3 Complex And CD2 ToActivate And Redirect Resting Cytotoxic T Cells,” J. Immunol. 147:60-69;U.S. Pat. Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and5,601,819; and Kostelny et al. (1992) “Formation Of A BispecificAntibody By The Use Of Leucine Zippers,” J. Immunol. 148:1547-1553;Todorovska et al. (2001) “Design And Application Of Diabodies,Triabodies And Tetrabodies For Cancer Targeting,” Journal ofImmunological Methods, 248:47-66. In particular embodiments, thehumanized antibodies of the invention are multispecific withspecificities for FcγRIIB and for a cancer antigen or any other cellsurface marker specific for a cell (e.g., an immune cell such as aT-cell or B-cell) designed to be killed, e.g., in treating or preventinga particular disease or disorder, or for other Fc receptors, e.g.,FcγRIIIA, FcγRIIIB, etc.

In a specific embodiment, an antibody used in the methods of the presentinvention is an antibody or an antigen-binding fragment thereof (e.g.,comprising one or more complementarily determining regions (CDRs),preferably all 6 CDRs) of the antibody produced by clone 2B6, 3H7, 1D5,2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively(e.g., the heavy chain CDR3). In another embodiment, an antibody used inthe methods of the present invention binds to the same epitope as themouse monoclonal antibody produced from clone 2B6, 3H7, 1D5, 2E1, 2H9,2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively and/or competeswith the mouse monoclonal antibody produced from clone 2B6, 3H7, 1D5,2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively asdetermined, e.g., in an ELISA assay or other appropriate competitiveimmunoassay, and also binds FcγRIIB with a greater affinity than saidantibody or a fragment thereof binds FcγRIIA.

The humanized antibodies used in the methods of the invention includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the antibody such that covalent attachment. Forexample, but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited to,specific chemical cleavage, acetylation, formylation, metabolicsynthesis of tunicamycin, etc. Additionally, the derivative may containone or more non-classical amino acids.

For some uses, including in vivo use of humanized antibodies in humansand in vitro detection assays, it may be preferable to use human,chimeric or humanized antibodies. Completely human antibodies areparticularly desirable for therapeutic treatment of human subjects.Human antibodies can be made by a variety of methods known in the artincluding phage display methods described above using antibody librariesderived from human immunoglobulin sequences. See also U.S. Pat. Nos.4,444,887 and 4,716,111; and International Publication Nos. WO 98/46645,WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741; each of which is incorporated herein by reference in itsentirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized using conventional methodologies with a selected antigen,e.g., all or a portion of a polypeptide of the invention. Monoclonalantibodies directed against the antigen can be obtained from theimmunized, transgenic mice using conventional hybridoma technology. Thehuman immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg et al. (1995) “Human antibodies from transgenicmice,” Int. Rev. Immunol. 13:65-93, which is incorporated herein byreference in its entirety. For a detailed discussion of this technologyfor producing human antibodies and human monoclonal antibodies andprotocols for producing such antibodies, see, e.g., InternationalPublication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S.Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016,5,545,806, 5,814,318, and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Medarex (Princeton, N.J.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. (e.g., Morrison (1985)“Transfectomas Provide Novel Chimeric Antibodies,” Science229:1202-1207; Oi et al. (1986) “Chimeric Antibodies,” BioTechniques4:214-221; Gillies et al. (1989) “High-Level Expression Of ChimericAntibodies Using Adapted cDNA Variable Region Cassettes,” J. Immunol.Methods 125:191-202; and U.S. Pat. Nos. 6,311,415, 5,807,715, 4,816,567,and 4,816,397, which are incorporated herein by reference in theirentirety. Chimeric antibodies comprising one or more CDRs from anon-human species and framework regions from a human immunoglobulinmolecule can be produced using a variety of techniques known in the artincluding, for example, CDR-grafting (EP 239,400; InternationalPublication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101,and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan(1991) “A Possible Procedure For Reducing The Immunogenicity Of AntibodyVariable Domains While Preserving Their Ligand-Binding Properties,”Molecular Immunology 28(4/5):489-498; Studnicka et al. (1994)“Human-Engineered Monoclonal Antibodies Retain Full Specific BindingActivity By Preserving Non-Cdr Complementarity-Modulating Residues,”Protein Engineering 7:805-814; and Roguska et al. (1994) “HumanizationOf Murine Monoclonal Antibodies Through Variable Domain Resurfacing,”Proc. Nat. Acad. Sci. 91:969-973), and chain shuffling (U.S. Pat. No.5,565,332). Each of the above-identified references is incorporatedherein by reference in its entirety.

Further, the antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies using techniques well known to thoseskilled in the art. (See, e.g., Greenspan et al. (1993) “Idiotypes:Structure And Immunogenicity,” FASEB J. 7:437-444; and Nissinoff (1991)“Idiotypes: Concepts And Applications,” J. Immunol. 147:2429-2438). Theinvention provides methods employing the use of polynucleotidescomprising a nucleotide sequence encoding an antibody of the inventionor a fragment thereof.

The present invention encompasses single domain antibodies, includingcamelized single domain antibodies (See e.g., Muyldermans et al. (2001)“Recognition Of Antigens By Single-Domain Antibody Fragments: TheSuperfluous Luxury Of Paired Domains,” Trends Biochem. Sci. 26:230-235;Nuttall et al. (2000), “Immunoglobulin VH Domains And Beyond: Design AndSelection Of Single-Domain Binding And Targeting Reagents,” Cur. Pharm.Biotech. 1:253-263; Reichmann et al. (1999) “Single Domain Antibodies:Comparison Of Camel VH And Camelised Human VH Domains,” J. Immunol.Meth. 231:25-38; International Publication Nos. WO 94/04678 and WO94/25591; U.S. Pat. No. 6,005,079; which are incorporated herein byreference in their entireties). In one embodiment, the present inventionprovides single domain antibodies comprising two VH domains withmodifications such that single domain antibodies are formed.

The methods of the present invention also encompass the use of humanizedantibodies or fragments thereof that have half-lives (e.g., serumhalf-lives) in a mammal, preferably a human, of greater than 15 days,preferably greater than 20 days, greater than 25 days, greater than 30days, greater than 35 days, greater than 40 days, greater than 45 days,greater than 2 months, greater than 3 months, greater than 4 months, orgreater than 5 months. The increased half-lives of the humanizedantibodies of the present invention or fragments thereof in a mammal,preferably a human, results in a higher serum titer of said antibodiesor antibody fragments in the mammal, and thus, reduces the frequency ofthe administration of said antibodies or antibody fragments and/orreduces the concentration of said antibodies or antibody fragments to beadministered. Antibodies or fragments thereof having increased in vivohalf-lives can be generated by techniques known to those of skill in theart. For example, antibodies or fragments thereof with increased in vivohalf-lives can be generated by modifying (e.g., substituting, deletingor adding) amino acid residues identified as involved in the interactionbetween the Fc domain and the FcRn receptor. The humanized antibodies ofthe invention may be engineered by methods described in Ward et al. toincrease biological half-lives (See U.S. Pat. No. 6,277,375 B1). Forexample, humanized antibodies of the invention may be engineered in theFc-hinge domain to have increased in vivo or serum half-lives.

Antibodies or fragments thereof with increased in vivo half-lives can begenerated by attaching to said antibodies or antibody fragments polymermolecules such as high molecular weight polyethyleneglycol (PEG). PEGcan be attached to said antibodies or antibody fragments with or withouta multifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of said antibodies or antibody fragments orvia epsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

The humanized antibodies of the invention may also be modified by themethods and coupling agents described by Davis et al. (See U.S. Pat. No.4,179,337) in order to provide compositions that can be injected intothe mammalian circulatory system with substantially no immunogenicresponse.

The present invention also encompasses the use of humanized antibodiesor antibody fragments comprising the amino acid sequence of any of theantibodies of the invention with mutations (e.g., one or more amino acidsubstitutions) in the framework or CDR regions. Preferably, mutations inthese humanized antibodies maintain or enhance the avidity and/oraffinity of the antibodies for CD32B to which they immunospecificallybind. Standard techniques known to those skilled in the art (e.g.,immunoassays) can be used to assay the affinity of an antibody for aparticular antigen.

The invention encompasses modification of framework residues of thehumanized antibodies of the invention. Framework residues in theframework regions may be substituted with the corresponding residue fromthe CDR donor antibody to alter, preferably improve, antigen binding.These framework substitutions are identified by methods well known inthe art, e.g., by modeling of the interactions of the CDR and frameworkresidues to identify framework residues important for antigen bindingand sequence comparison to identify unusual framework residues atparticular positions. (See, e.g., U.S. Pat. No. 5,585,089; and Riechmannet al. (1988) “Reshaping Human Antibodies For Therapy,” Nature332:323-327, which are incorporated herein by reference in theirentireties.)

The present invention encompasses humanized antibodies comprisingmodifications preferably, in the Fc region that modify the bindingaffinity of the antibody to one or more FcγR. Methods for modifyingantibodies with modified binding to one or more FcγR are known in theart (see, e.g., PCT Publication Nos. WO 04/029207, WO 04/029092, WO04/028564, WO 99/58572, WO 99/51642, WO 98/23289, WO 89/07142, WO88/07089, and U.S. Pat. Nos. 5,843,597 and 5,642,821, each of which isincorporated herein by reference in their entirety). The inventionencompasses any of the mutations disclosed in U.S. Application Nos.60/439,498 and 60/456,041, filed Jan. 9, 2003 and Mar. 19, 2003,respectively each of which is incorporated herein by reference in theirentirety. In some embodiments, the invention encompasses antibodies thathave altered affinity for an activating FcγR, e.g., FcγRIIIA. Preferablysuch modifications also have an altered Fc-mediated effector function.Modifications that affect Fc-mediated effector function are well knownin the art (See U.S. Pat. No. 6,194,551, which is incorporated herein byreference in its entirety). The amino acids that can be modified inaccordance with the method of the invention include, but are not limitedto, Proline 329, Proline 331, and Lysine 322. Proline 329, Proline 331and Lysine 322 are preferably replaced with alanine, however,substitution with any other amino acid is contemplated. (SeeInternational Publication No.: WO 00/42072 and U.S. Pat. No. 6,194,551which are incorporated herein by reference in their entirety).

In one particular embodiment, the modification of the Fc regioncomprises one or more mutations in the Fc region. The one or moremutations in the Fc region may result in an antibody with an alteredantibody-mediated effector function, an altered binding to other Fcreceptors (e.g., Fc activation receptors), an altered ADCC activity, analtered C1q binding activity, an altered complement dependentcytotoxicity activity, a phagocytic activity, or any combinationthereof.

The invention also provides humanized antibodies with alteredoligosaccharide content. Oligosaccharides, as used herein, refer tocarbohydrates containing two or more simple sugars and the two terms maybe used interchangeably herein. Carbohydrate moieties of the instantinvention will be described with reference to commonly used nomenclaturein the art. For a review of carbohydrate chemistry see Hubbard et al.(1981) “Synthesis and Processing of Asparagine-Linked Oligosaccharides,”Ann. Rev. Biochem., 50: 555-583, which is incorporated herein byreference in its entirety. This nomenclature includes, for example, Manwhich represents mannose; GlcNAc which represents 2-N-acetylglucosamine;Gal which represents galactose; Fuc for fucose and Glc for glucose.Sialic acids are described by the shorthand notation NeuNAc for5-N-acetylneuraminic acid, and NeuNGc for 5-glycolneuraminic.

In general, antibodies contain carbohydrate moeities at conservedpositions in the constant region of the heavy chain, and up to 30% ofhuman IgGs have a glycosylated Fab region. IgG has a single N-linkedbiantennary carbohydrate structure at Asn 297 which resides in the CH2domain (Jefferis et al. (1998) “IgG-Fc-Mediated Effector Functions:Molecular Definition Of Interaction Sites For Effector Ligands And TheRole Of Glycosylation,” Immunol. Rev. 163: 59-76; Wright et al. (1997)“Effect Of Glycosylation On Antibody Function: Implications For GeneticEngineering,” Trends Biotech 15: 26-32). Human IgG typically has acarbohydrate of the following structure;GlcNAc(Fucose)-GlcNAc-Man-(ManGlcNAc)₂. However variations among IgGs incarbohydrate content does occur which leads to altered function (e.g.,Jassal et al. (2001) “Sialylation Of Human IgG-Fc Carbohydrate ByTransfected Rat Alpha2,6-sialyltransferase,” Biochem. Biophys. Res.Commun. 288: 243-249; Groenink et al. (1996) “On The Interaction BetweenAgalactosyl IgG And Fc Gamma Receptors,” Eur. J. Immunol. 26: 1404-1407;Boyd et al. (1995) “The Effect Of The Removal Of Sialic Acid, GalactoseAnd Total Carbohydrate On The Functional Activity Of Campath-1H,” Mol.Immunol. 32: 1311-1318; Kumpel et al. (1994) “Galactosylation Of HumanIgG Monoclonal Anti-D Produced By EBV-Transformed B-lymphoblastoid CellLines Is Dependent On Culture Method And Affects Fc Receptor-MediatedFunctional Activity,” Human Antibody Hybridomas 5: 143-151. Theinvention encompasses humanized antibodies comprising a variation in thecarbohydrate moiety that is attached to Asn 297. In one embodiment, thecarbohydrate moiety has a galactose and/or galactose-sialic acid at oneor both of the terminal GlcNAc and/or a third GlcNac arm (bisectingGlcNAc).

In some embodiments, the humanized antibodies of the invention aresubstantially free of one or more selected sugar groups, e.g., one ormore sialic acid residues, one or more galactose residues, one or morefucose residues. An antibody that is substantially free of one or moreselected sugar groups may be prepared using common methods known to oneskilled in the art, including, for example, recombinantly producing anantibody of the invention in a host cell that is defective in theaddition of the selected sugar groups(s) to the carbohydrate moiety ofthe antibody, such that about 90-100% of the antibody in the compositionlacks the selected sugar group(s) attached to the carbohydrate moiety.Alternative methods for preparing such antibodies include, for example,culturing cells under conditions which prevent or reduce the addition ofone or more selected sugar groups, or post-translational removal of oneor more selected sugar groups.

In a specific embodiment, the invention encompasses a method ofproducing a substantially homogenous antibody preparation, wherein about80-100% of the antibody in the composition lacks a fucose on itscarbohydrate moiety. The antibody may be prepared, for example, by (A)use of an engineered host cell that is deficient in fucose metabolismsuch that it has a reduced ability to fucosylate proteins expressedtherein; (B) culturing cells under conditions which prevent or reducefusocylation; (C) post-translational removal of fucose, e.g., with afucosidase enzyme; or (D) purification of the antibody so as to selectfor the product which is not fucosylated. Most preferably, a nucleicacid encoding the desired antibody is expressed in a host cell that hasa reduced ability to fucosylate the antibody expressed therein.Preferably, the host cell is a Lec 13 CHO cell (lectin resistant CHOmutant cell line; U.S. Patent Application Publication No. 2003/0115614;PCT Publication No. WO 00/61739; European Patent Application EP 1 229125; Ripka et al. (1986) “Lectin-Resistant CHO Cells: Selection Of FourNew Pea Lectin-Resistant Phenotypes,” Somatic Cell & Molec. Gen.12:51-62; Ripka et al. (1986) “Two Chinese Hamster Ovary GlycosylationMutants Affected In The Conversion Of GDP-Mannose To GDP-Fucose,” Arch.Biochem. Biophys. 249:533-545), CHO-K1 cell, DUX-B11 cell, CHO-DP12 cellor CHO-DG44 cell, which has been modified so that the antibody is notsubstantially fucosylated. Thus, the cell may display altered expressionand/or activity for the fucoysltransferase enzyme, or another enzyme orsubstrate involved in adding fucose to the N-linked oligosaccharide sothat the enzyme has a diminished activity and/or reduced expressionlevel in the cell. For methods to produce antibodies with altered fucosecontent (e.g., WO 03/035835 and Shields et al. (2002) “Lack Of Fucose OnHuman IgG1 N-linked Oligosaccharide Improves Binding To Human FcgammaRIII And Antibody-Dependent Cellular Toxicity,” J. Biol. Chem.277:26733-26740; both of which are incorporated herein by reference intheir entirety).

In some embodiments, the altered carbohydrate modifications modulate oneor more of the following: solubilization of the antibody, facilitationof subcellular transport and secretion of the antibody, promotion ofantibody assembly, conformational integrity, and antibody-mediatedeffector function. In a specific embodiment the altered carbohydratemodifications enhance antibody mediated effector function relative tothe antibody lacking the carbohydrate modification. Carbohydratemodifications that lead to altered antibody mediated effector functionare well known in the art (for example, see Shields et al. (2002) “LackOf Fucose On Human IgG1 N-linked Oligosaccharide Improves Binding ToHuman Fcgamma RIII And Antibody-Dependent Cellular Toxicity,” J. Biol.Chem. 277:26733-26740; Davies J. et al. (2001) “Expression Of GnTIII InA Recombinant Anti-CD20 CHO Production Cell Line: Expression OfAntibodies With Altered Glycoforms Leads To An Increase In ADCC ThroughHigher Affinity For FC gamma RIII,” Biotechnology & Bioengineering, 74:288-294). In another specific embodiment, the altered carbohydratemodifications enhance the binding of antibodies of the invention toFcγRIIB receptor. Altering carbohydrate modifications in accordance withthe methods of the invention includes, for example, increasing thecarbohydrate content of the antibody or decreasing the carbohydratecontent of the antibody. Methods of altering carbohydrate contents areknown to those skilled in the art, e.g., Wallick et al. (1988)“Glycosylation Of A VH Residue Of A Monoclonal Antibody Against Alpha(1 - - - 6) Dextran Increases Its Affinity For Antigen,” Journal of Exp.Med. 168:1099-1109; Tao et al. (1989) “Studies Of Aglycosylated ChimericMouse-Human IgG. Role Of Carbohydrate In The Structure And EffectorFunctions Mediated By The Human IgG Constant Region,” Journal ofImmunology, 143:2595-2601; Routledge et al. (1995) “The Effect OfAglycosylation On The Immunogenicity Of A Humanized Therapeutic CD3Monoclonal Antibody,” Transplantation, 60:847-53; Elliott et al. (2003)“Enhancement Of Therapeutic Protein In Vivo Activities ThroughGlycoengineering,” Nature Biotechnology, 21: 414-21; Shields et al.(2002) “Lack Of Fucose On Human IgG1 N-linked Oligosaccharide ImprovesBinding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity,”J. Biol. Chem. 277:26733-26740; all of which are incorporated herein byreference in their entirety.

In some embodiments, the invention encompasses humanized antibodiescomprising one or more glycosylation sites, so that one or morecarbohydrate moieties are covalently attached to the antibody. In otherembodiments, the invention encompasses humanized antibodies comprisingone or more glycosylation sites and one or more modifications in the Fcregion, such as those disclosed supra and those known to one skilled inthe art. In preferred embodiments, the one or more modifications in theFc region enhance the affinity of the antibody for an activating FcγR,e.g., FcγRIIIA, relative to the antibody comprising the wild type Fcregions. Humanized antibodies of the invention with one or moreglycosylation sites and/or one or more modifications in the Fc regionhave an enhanced antibody mediated effector function, e.g., enhancedADCC activity. In some embodiments, the invention further compriseshumanized antibodies comprising one or more modifications of amino acidsthat are directly or indirectly known to interact with a carbohydratemoiety of the antibody, including, but not limited to, amino acids atpositions 241, 243, 244, 245, 245, 249, 256, 258, 260, 262, 264, 265,296, 299, and 301. Amino acids that directly or indirectly interact witha carbohydrate moiety of an antibody are known in the art, see, e.g.,Jefferis et al. (1995) “Recognition Sites On Human IgG For Fc gammaReceptors: The Role Of Glycosylation,” Immunology Letters, 44: 111-117,which is incorporated herein by reference in its entirety.

The invention encompasses humanized antibodies that have been modifiedby introducing one or more glycosylation sites into one or more sites ofthe antibodies, preferably without altering the functionality of theantibody, e.g., binding activity to FcγRIIB. Glycosylation sites may beintroduced into the variable and/or constant region of the antibodies ofthe invention. As used herein, “glycosylation sites” include anyspecific amino acid sequence in an antibody to which an oligosaccharide(i.e., carbohydrates containing two or more simple sugars linkedtogether) will specifically and covalently attach. Oligosaccharide sidechains are typically linked to the backbone of an antibody via either N-or O-linkages. N-linked glycosylation refers to the attachment of anoligosaccharide moiety to the side chain of an asparagine residue.O-linked glycosylation refers to the attachment of an oligosaccharidemoiety to a hydroxyamino acid, e.g., serine, threonine. The antibodiesof the invention may comprise one or more glycosylation sites, includingN-linked and O-linked glycosylation sites. Any glycosylation site forN-linked or O-linked glycosylation known in the art may be used inaccordance with the instant invention. An exemplary N-linkedglycosylation site that is useful in accordance with the methods of thepresent invention, is the amino acid sequence: Asn-X-Thr/Ser, wherein Xmay be any amino acid and Thr/Ser indicates a threonine or a serine.Such a site or sites may be introduced into an antibody of the inventionusing methods well known in the art to which this invention pertains.(“In Vitro Mutagenesis,” RECOMBINANT DNA: A SHORT COURSE, J. D. Watson,et al. W.H. Freeman and Company, New York, 1983, chapter 8, pp. 106-116,which is incorporated herein by reference in its entirety.) An exemplarymethod for introducing a glycosylation site into an antibody of theinvention may comprise: modifying or mutating an amino acid sequence ofthe antibody so that the desired Asn-X-Thr/Ser sequence is obtained.

In some specific embodiments, the invention encompasses modifiedhumanized FcγRIIB antibodies wherein the N-glysosylation consensenussite Asn₅₀-Val-Ser of the CDR2 region has been modified, so that theglycosylation site at position 50 is eliminated. Although not intendingto be bound by a particular mechanism of action, removal of theglycosylation site may limit potential variation in production of theantibody as well as potential immunogenicity in a pharmaceuticalapplication. In a specific embodiment, the invention encompasses ahumanized FcγRIIB antibody wherein the amino acid at position 50 hasbeen modified, e.g., deleted or substituted. In another specificembodiment, the invention further encompasses an amino acidmodification, e.g., deletion or substitution, at position 51. In onespecific embodiment, the invention encompasses a humanized FcγRIIBantibody wherein the amino acid at position 50 has been replaced withtyrosine. In another more specific embodiment, the invention encompassesa humanized FcγRIIB antibody wherein the amino acid at position 50 hasbeen replaced with tyrosine and the amino acid at position 51 has beenreplaced with alanine.

In some embodiments, the invention encompasses methods of modifying thecarbohydrate content of an antibody of the invention by adding ordeleting a glycosylation site. Methods for modifying the carbohydratecontent of antibodies are well known in the art and encompassed withinthe invention, see, e.g., U.S. Pat. No. 6,218,149; EP 0 359 096 B1; U.S.Patent Application Publication No. US 2002/0028486; WO 03/035835; U.S.Publication No. 2003/0115614; U.S. Pat. No. 6,218,149; U.S. Pat. No.6,472,511; all of which are incorporated herein by reference in theirentirety. In other embodiments, the invention encompasses methods ofmodifying the carbohydrate content of an antibody of the invention bydeleting one or more endogenous carbohydrate moieties of the antibody.

The invention further encompasses methods of modifying an effectorfunction of an antibody of the invention, wherein the method comprisesmodifying the carbohydrate content of the antibody using the methodsdisclosed herein or known in the art.

Standard techniques known to those skilled in the art can be used tointroduce mutations in the nucleotide sequence encoding an antibody, orfragment thereof, including, e.g., site-directed mutagenesis andPCR-mediated mutagenesis, which results in amino acid substitutions.Preferably, the derivatives include less than 15 amino acidsubstitutions, less than 10 amino acid substitutions, less than 5 aminoacid substitutions, less than 4 amino acid substitutions, less than 3amino acid substitutions, or less than 2 amino acid substitutionsrelative to the original antibody or fragment thereof. In a preferredembodiment, the derivatives have conservative amino acid substitutionsmade at one or more predicted non-essential amino acid residues.

The present invention also encompasses humanized antibodies or fragmentsthereof comprising an amino acid sequence of a variable heavy chainand/or variable light chain that is at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 99% identicalto the amino acid sequence of the variable heavy chain and/or lightchain of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5,2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively. Thepresent invention further encompasses antibodies or fragments thereofthat specifically bind FcγRIIB with greater affinity than said antibodyor fragment thereof binds FcγRIIA, said antibodies or antibody fragmentscomprising an amino acid sequence of one or more CDRs that is at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 99% identical to the amino acid sequence of one or moreCDRs of the mouse monoclonal antibody produced by clone 2B6, 3H7, 1D5,2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592,PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively. Thedetermination of percent identity of two amino acid sequences can bedetermined by any method known to one skilled in the art, includingBLAST protein searches.

The present invention also encompasses the use of humanized antibodiesor antibody fragments that specifically bind FcγRIIB with greateraffinity than said antibodies or fragments thereof binds FcγRIIA,wherein said antibodies or antibody fragments are encoded by anucleotide sequence that hybridizes to the nucleotide sequence of themouse monoclonal antibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9,2D11, or 1F2, with ATCC accession numbers PTA-4591, PTA-4592, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively, understringent conditions. In a preferred embodiment, the invention providesantibodies or fragments thereof that specifically bind FcγRIIB withgreater affinity than said antibodies or fragments thereof bind FcγRIIA,said antibodies or antibody fragments comprising a variable light chainand/or variable heavy chain encoded by a nucleotide sequence thathybridizes under stringent conditions to the nucleotide sequence of thevariable light chain and/or variable heavy chain of the mouse monoclonalantibody produced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, withATCC accession numbers PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively, under stringent conditions. Inanother preferred embodiment, the invention provides antibodies orfragments thereof that specifically bind FcγRIIB with greater affinitythan said antibodies or fragments thereof bind FcγRIIA, said antibodiesor antibody fragments comprising one or more CDRs encoded by anucleotide sequence that hybridizes under stringent conditions to thenucleotide sequence of one or more CDRs of the mouse monoclonal antibodyproduced by clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, with ATCCaccession numbers PTA-4591, PTA-4592, PTA-5958, PTA-5961, PTA-5962,PTA-5960, and PTA-5959, respectively. Stringent hybridization conditionsinclude, but are not limited to, hybridization to filter-bound DNA in 6×sodium chloride/sodium citrate (SSC) at about 45° C. followed by one ormore washes in 0.2×SSC/0.1% SDS at about 50-65° C., highly stringentconditions such as hybridization to filter-bound DNA in 6×SSC at about45° C. followed by one or more washes in 0.1×SSC/0.2% SDS at about 60°C., or any other stringent hybridization conditions known to thoseskilled in the art (see, for example, Ausubel et al., eds. 1989 CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, vol. 1, Green Publishing Associates,Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to 6.3.6 and2.10.3, incorporated herein by reference).

II. Antibody Conjugates

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to heterologous polypeptides (i.e., an unrelatedpolypeptide; or portion thereof, preferably at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids of the polypeptide) togenerate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. Antibodies may be usedfor example to target heterologous polypeptides to particular celltypes, either in vitro or in vivo, by fusing or conjugating theantibodies to antibodies specific for particular cell surface receptors.Antibodies fused or conjugated to heterologous polypeptides may also beused in in vitro immunoassays and purification methods using methodsknown in the art. See e.g., PCT Publication No. WO 93/21232; EP 439,095;Naramura et al. (1994) “Mechanisms Of Cellular Cytotoxicity Mediated ByA Recombinant Antibody-IL2 Fusion Protein Against Human Melanoma Cells,”Immunol. Lett., 39:91-99; U.S. Pat. No. 5,474,981; Gillies et al. (1992)“Antibody-Targeted Interleukin 2 Stimulates T-Cell Killing Of AutologousTumor Cells,” Proc. Nat. Acad. Sci., 89:1428-1432; and Fell et al.(1991) “Genetic Construction And Characterization Of A Fusion ProteinConsisting Of A Chimeric F(ab′) With Specificity For Carcinomas AndHuman IL-2,” J. Immunol., 146:2446-2452, each of which is incorporatedherein by reference in their entireties.

Further, a humanized antibody may be conjugated to a therapeutic agentor drug moiety that modifies a given biological response. Therapeuticagents or drug moieties are not to be construed as limited to classicalchemical therapeutic agents. For example, the drug moiety may be aprotein or polypeptide possessing a desired biological activity. Suchproteins may include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin (i.e., PE-40), or diphtheria toxin, ricin, gelonin,and pokeweed antiviral protein, a protein such as tumor necrosis factor,interferons including, but not limited to, α-interferon (IFN-α),β-interferon (IFN-β), nerve growth factor (NGF), platelet derived growthfactor (PDGF), tissue plasminogen activator (TPA), an apoptotic agent(e.g., TNF-α, TNF-β, AIM I as disclosed in PCT Publication No. WO97/33899), AIM II (see, e.g., PCT Publication No. WO 97/34911), FasLigand (Takahashi et al. (1994) “Human Fas Ligand: Gene Structure,Chromosomal Location And Species Specificity,” Int. Immunol.,6:1567-1574), and VEGI (PCT Publication No. WO 99/23105), a thromboticagent or an anti-angiogenic agent (e.g., angiostatin or endostatin), ora biological response modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), macrophage colonystimulating factor, (“M-CSF”), or a growth factor (e.g., growth hormone(“GH”); a protease, or a ribonuclease.

Humanized antibodies can be fused to marker sequences, such as apeptide, to facilitate purification. In preferred embodiments, themarker amino acid sequence is a hexa-histidine peptide, such as the tagprovided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311), among others, many of which are commercially available.As described in Gentz et al. (1989) “Bioassay For Trans-Activation UsingPurified Human Immunodeficiency Virus Tat-Encoded Protein:Trans-Activation Requires mRNA Synthesis,” Proc. Natl. Acad. Sci. USA,86:821-824, for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the hemagglutinin “HA”tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al. (1984) “The Structure Of AnAntigenic Determinant In A Protein,” Cell, 37:767-778) and the “flag”tag (Knappik et al. (1994) “An Improved Affinity Tag Based On The FLAGPeptide For The Detection And Purification Of Recombinant AntibodyFragments,” Biotechniques, 17(4):754-761).

The present invention further includes the use of compositionscomprising heterologous polypeptides fused or conjugated to antibodyfragments. For example, the heterologous polypeptides may be fused orconjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)₂ fragment,or portion thereof. Methods for fusing or conjugating polypeptides toantibody portions are known in the art. See, e.g., U.S. Pat. Nos.5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EP307,434; EP 367,166; International Publication Nos. WO 96/04388 and WO91/06570; Ashkenazi et al. (1991) “Protection Against Endotoxic Shock ByA Tumor Necrosis Factor Receptor Immunoadhesin,” Proc. Nat. Acad. Sci.88: 10535-10539; Zheng et al. (1995) “Administration Of NoncytolyticIL-10/Fc In Murine Models Of Lipopolysaccharide-Induced Septic Shock AndAllogeneic Islet Transplantation,” J. Immunol. 154:5590-5600; and Vie etal. (1992) “Human Fusion Proteins Between Interleukin 2 And IgM HeavyChain Are Cytotoxic For Cells Expressing The Interleukin 2 Receptor,”Proc. Nat. Acad. Sci. 89:11337-11341 (said references incorporated byreference in their entireties).

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten etal. (1997) “Applications Of DNA Shuffling To Pharmaceuticals AndVaccines,” Curr. Opinion Biotechnol. 8:724-733; Harayama (1998)“Artificial Evolution By DNA Shuffling,” Trends Biotechnol. 16:76-82;Hansson, et al. (1999) “Evolution Of Differential SubstrateSpecificities In Mu Class Glutathione Transferases Probed By DNAShuffling,” J. Mol. Biol. 287:265-276; and Lorenzo et al. (1998)“PCR-Based Method For The Introduction Of Mutations In Genes Cloned AndExpressed In Vaccinia Virus,” BioTechniques 24:308-313 (each of thesepatents and publications are hereby incorporated by reference in itsentirety). Antibodies or fragments thereof, or the encoded antibodies orfragments thereof, may be altered by being subjected to randommutagenesis by error-prone PCR, random nucleotide insertion or othermethods prior to recombination. One or more portions of a polynucleotideencoding an antibody or antibody fragment, which portions specificallybind to FcγRIIB may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

The present invention also encompasses humanized antibodies conjugatedto a diagnostic or therapeutic agent or any other molecule for whichserum half-life is desired to be increased. The humanized antibodies canbe used diagnostically to, for example, monitor the development orprogression of a disease, disorder or infection as part of a clinicaltesting procedure to, e.g., determine the efficacy of a given treatmentregimen. Detection can be facilitated by coupling the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. See, forexample, U.S. Pat. No. 4,741,900 for metal ions which can be conjugatedto antibodies for use as diagnostics according to the present invention.Such diagnosis and detection can be accomplished by coupling theantibody to detectable substances including, but not limited to, variousenzymes, enzymes including, but not limited to, horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;prosthetic group complexes such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent material such as, but not limitedto, luminol; bioluminescent materials such as, but not limited to,luciferase, luciferin, and aequorin; radioactive material such as, butnot limited to, bismuth (²¹³Bi), carbon (¹⁴C), chromium (⁵¹Cr), cobalt(⁵⁷Co), fluorine (¹⁸F), gadolinium (¹⁵³Gd, ¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga),germanium (⁶⁸Ge), holmium (¹⁶⁶Ho), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), lanthanium (¹⁴⁰La), lutetium (¹⁷⁷Lu),manganese (⁵⁴Mn), molybdenum (⁹⁹Mo), palladium (¹⁰³Pd), phosphorous(³²P), praseodymium (¹⁴²Pr), promethium (¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re),rhodium (¹⁰⁵Rh), ruthemium (⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc),selenium (⁷⁵Se), strontium (⁸⁵Sr), sulfur (³⁵S), technetium (⁹⁹Tc),thallium (²⁰¹Ti), tin (¹¹³Sn, ¹¹⁷Sn), tritium (³H), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb), yttrium (⁹⁰Y), zinc (⁶⁵ Zn); positron emittingmetals using various positron emission tomographies, and nonradioactiveparamagnetic metal ions.

An antibody may be conjugated to a therapeutic moiety such as acytotoxin (e.g., a cytostatic or cytocidal agent), a therapeutic agentor a radioactive element (e.g., alpha-emitters, gamma-emitters, etc.).Cytotoxins or cytotoxic agents include any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, BiCNU® (carmustine; BSNU) and lomustine(CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin,mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Moreover, a humanized antibody can be conjugated to therapeutic moietiessuch as a radioactive materials or macrocyclic chelators useful forconjugating radiometal ions (see above for examples of radioactivematerials). In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.(1998) “Comparison Of1,4,7,10-Tetraazacyclododecane-N,N′,N″,N′″-Tetraacetic Acid(DOTA)-Peptide-ChL6, A Novel Immunoconjugate With Catabolizable Linker,To 2-Iminothiolane-2-[p-(bromoacetamido)benzyl]-DOTA-ChL6 In BreastCancer Xenografts,” Clin. Cancer Res. 4:2483-2490; Peterson et al.(1999) “Enzymatic Cleavage Of Peptide-Linked Radiolabels FromImmunoconjugates,” Bioconjug. Chem. 10:553-557; and Zimmerman et al.(1999) “A Triglycine Linker Improves Tumor Uptake And BiodistributionsOf 67-Cu-Labeled Anti-Neuroblastoma MAb chCE7 F(ab′)2 Fragments,” Nucl.Med. Biol. 26:943-950, each incorporated by reference in theirentireties.

Techniques for conjugating such therapeutic moieties to antibodies arewell known; see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in MONOCLONAL ANTIBODIESAND CANCER THERAPY, Reisfeld et al. (eds.), 1985, pp. 243-256, Alan R.Liss, Inc.); Hellstrom et al., “Antibodies For Drug Delivery”, inCONTROLLED DRUG DELIVERY (2nd Ed.), Robinson et al. (eds.), 1987, pp.623-53, Marcel Dekker, Inc.); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in MONOCLONAL ANTIBODIES '84:BIOLOGICAL AND CLINICAL APPLICATIONS, Pinchera et al. (eds.), 1985, pp.475-506); “Analysis, Results, And Future Prospective Of The TherapeuticUse Of Radiolabeled Antibody In Cancer Therapy”, in MONOCLONALANTIBODIES FOR CANCER DETECTION AND THERAPY, Baldwin et al. (eds.),1985, pp. 303-16, Academic Press; and Thorpe et al. (1982) “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates,”Immunol. Rev., 62:119-158.

An antibody or fragment thereof, with or without a therapeutic moietyconjugated to it, administered alone or in combination with cytotoxicfactor(s) and/or cytokine(s) can be used as a therapeutic.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

III. Preparation of FcγRIIB Humanized Antibodies

The invention encompasses nucleotide sequences that encode theCDR-grafted heavy and light chains, cloning and expression vectorscontaining the nucleotide sequences, host cells transformed with thenucleotide sequences, and methods for the production of the CDR-graftedchains and antibody molecules comprising the nucleotide sequences in thetransformed host cells. In specific embodiments, the inventionencompasses the nucleotide sequences of:

(SEQ ID NO.: 17) gaaattgtgc tgactcagtc tccagacttt cagtctgtga ctccaaaggagaaagtcacc atcacctgca ggaccagtca gagcattggc acaaacatac actggtaccagcagaaacca gatcagtctc caaagctcct catcaagaat gtttctgagt ctatctctggagtcccatcg aggttcagtg gcagtggatc tgggacagat ttcaccctca ccatcaatagcctggaagct gaagatgctg caacgtatta ctgtcaacaa agtaatacct ggccgttcacgttcggcgga gggaccaagg tggagatcaa a; or (SEQ ID NO:19) gaaattgtgctgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc atcacctgcaggaccagtca gagcattggc acaaacatac actggtacca gcagaaacca gatcagtctccaaagctcct catcaagtat gtttctgagt ctatctctgg agtcccatcg aggttcagtggcagtggatc tgggacagat ttcaccctca ccatcaatag cctggaagct gaagatgctgcaacgtatta ctgtcaacaa agtaatacct ggccgttcac gttcggcgga gggaccaaggtggagatcaa a; or (SEQ ID NO:21) gaaattgtgc tgactcagtc tccagactttcagtctgtga ctccaaagga gaaagtcacc atcacctgca ggaccagtca gagcattggcacaaacatac actggtacca gcagaaacca gatcagtctc caaagctcct catcaagtatgcttctgagt ctatctctgg agtcccatcg aggttcagtg gcagtggatc tgggacagatttcaccctca ccatcaatag cctggaagct gaagatgctg caacgtatta ctgtcaacaaagtaatacct ggccgttcac gttcggcgga gggaccaagg tggagatcaa a; or (SEQ IDNO:23) caggttcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc agtgaaggtctcctgcaagg cttctggtta cacctttacc aactactgga tacactgggt gcgacaggcccctggacaag ggcttgagtg gatgggagtg attgatcctt ctgatactta tccaaattacaataaaaagt tcaagggcag agtcaccatg accacagaca catccacgag cacagcctacatggagctga ggagcctgag atctgacgac acggccgtgt attactgtgc gagaaacggtgattccgatt attactctgg tatggactac tgggggcaag ggaccacggt caccgtctcc tca;or (SEQ ID NO:36) gaagtgaagt ttgaggagtc tggaggaggc ttggtgcaac ctggaggatccatgaaactc tcttgtgctg cctctggatt cacttttagt gacgcctgga tggactgggtccgccagggt ccagagaagg ggcttgagtg ggttgctgaa attagaaaca aagctaataatcttgcaaca tactatgctg agtctgtgaa agggaggttc accatcccaa gagatgattccaaaagtagt gtctacctgc acatgaacag cttaagagct gaagacactg gcatttattactgttatagt ccctttgctt actggggcca agggactctg gtcactgtct ctgca; or (SEQ IDNO:45) gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagtctcacttgtc gggcaagtca ggaaattagt ggttacttaa gctggcttca gcagaaaccagatggaacta ttagacgcct gatctacgcc gcatccactt tagattctgg tgtcccaaaaaggttcagtg gcagttggtc tgggtcagat tattctctca ccatcagcag ccttgagtctgaagattttg cagactatta ctgtctacaa tatgttagtt atccgtatac gttcggaggggggaccaagc tggaaataaa a.

The invention encompasses donor amino acid sequences, which encodeantibodies that bind FcγRIIB with a greater affinity that FcγRIIA, suchas those disclosed in U.S. Provisional Application No. 60/403,366, filedon Aug. 14, 2002 and U.S. Patent Application Publication No.2004/0185045, both of which are incorporated herein by reference intheir entireties. In a specific embodiment, the donor amino acidsequence encodes for the monoclonal antibody produced from clone 2B6,3H7, 1D5, 2E1, 2H9, 2D11, or 1F2, with ATCC accession numbers PTA-4591,PTA-4592, PTA-5958, PTA-5961, PTA-5962, PTA-5960, and PTA-5959,respectively, or other monoclonal antibodies produced by immunizationmethods of the invention as disclosed in U.S. Provisional ApplicationNo. 60/403,366, filed on Aug. 14, 2002 and U.S. Patent ApplicationPublication No. 2004/0185045, both of which are incorporated herein byreference in their entireties. The invention also encompasspolynucleotides that encode for donor amino acid sequences thathybridize under various stringency, e.g., high stringency, intermediateor low stringency conditions, to polynucleotides that encode for themonoclonal antibody produced from clone 2B6, 3H7, 1D5, 2E1, 2H9, 2D11,or 1F2, with ATCC accession numbers PTA-4591, PTA-4592, PTA-5958,PTA-5961, PTA-5962, PTA-5960, and PTA-5959, respectively, or othermonoclonal antibodies produced by immunization methods of the inventionas disclosed in U.S. Provisional Application No. 60/403,366, filed onAug. 14, 2002 and U.S. Patent Application Publication No. 2004/0185045.The hybridization can be performed under various conditions ofstringency. By way of example and not limitation, procedures usingconditions of low stringency are as follows (see also Shilo et al.(1981) “DNA Sequences Homologous To Vertebrate Oncogenes Are ConservedIn Drosophila Melanogaster,” Proc. Natl. Acad. Sci. U.S.A.78:6789-6792). Filters containing DNA are pretreated for 6 h at 40° C.in a solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5),5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmonsperm DNA. Hybridizations are carried out in the same solution with thefollowing modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/mlsalmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm³²P-labeled probe is used. Filters are incubated in hybridizationmixture for 18-20 h at 40° C., and then washed for 1.5 h at 55° C. in asolution containing 2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1%SDS. The wash solution is replaced with fresh solution and incubated anadditional 1.5 h at 60° C. Filters are blotted dry and exposed forautoradiography. If necessary, filters are washed for a third time at65-68° C. and re-exposed to film. Other conditions of low stringencywhich may be used are well known in the art (e.g., as employed forcross-species hybridizations). By way of example and not limitation,procedures using conditions of high stringency are as follows.Prehybridization of filters containing DNA is carried out for 8 h toovernight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/mldenatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C. inprehybridization mixture containing 100 μg/ml denatured salmon sperm DNAand 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters is done at 37°C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and0.01% BSA. This is followed by a wash in 0.1×SSC at 50° C. for 45 minbefore autoradiography. Other conditions of high stringency which may beused are well known in the art. Selection of appropriate conditions forsuch stringencies is well known in the art (e.g., Sambrook et al.,(1989) MOLECULAR CLONING, A LABORATORY MANUAL, 2d Ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; see also, Ausubel etal., eds., in the CURRENT PROTOCOLS IN MOLECULAR BIOLOGY series oflaboratory technique manuals, ® 1987-1997, Current Protocols, ®1994-1997 John Wiley and Sons, Inc.; see especially, Dyson, 1991,“Immobilization Of Nucleic Acids And Hybridization Analysis,” in:ESSENTIAL MOLECULAR BIOLOGY: A PRACTICAL APPROACH, Vol. 2, T. A. Brown,ed., pp. 111-156, IRL Press at Oxford University Press, Oxford, UK). Thepolynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art.

DNA sequences which encode the acceptor amino acid sequences may beobtained by any method known to one skilled in the art. For example, DNAsequences coding for preferred human acceptor framework sequencesinclude but are not limited to FR segments from the human germline VHsegment VH1-8 and JH6 and the human germline VL segment VK-A26 and JK4.

In a specific embodiment, one or more of the CDRs are inserted withinframework regions using routine recombinant DNA techniques. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (e.g., Chothia et al.(1998) “Structural Determinants In The Sequences Of ImmunoglobulinVariable Domain,” J. Mol. Biol. 278:457-479 for a listing of humanframework regions). Preferably, the polynucleotide generated by thecombination of the framework regions and CDRs encodes an antibody thatspecifically binds to FcγRIIB with greater affinity than said antibodybinds FcγRIIA. Preferably, as discussed supra, one or more amino acidsubstitutions may be made within the framework regions, and, preferably,the amino acid substitutions improve binding of the antibodies of theinvention to FcγRIIB.

In another embodiment, human libraries or any other libraries availablein the art, can be screened by standard techniques known in the art, toclone the nucleic acids encoding the antibodies of the invention.

The humanized antibodies of the present invention may be produced by anymethod known in the art useful for the production of polypeptides, e.g.,in vitro synthesis, recombinant DNA production, and the like.Preferably, the humanized antibodies are produced by recombinant DNAtechnology. The humanized FcγRIIB-specific antibodies of the inventionmay be produced using recombinant immunoglobulin expression technology.The recombinant production of immunoglobulin molecules, includinghumanized antibodies are described in U.S. Pat. No. 4,816,397 (Boss etal.), U.S. Pat. Nos. 6,331,415 and 4,816,567 (both to Cabilly et al.),U.K. patent GB 2,188,638 (Winter et al.), and U.K. patent GB 2,209,757;all of which are incorporated herein by reference in their entireties.Techniques for the recombinant expression of immunoglobulins, includinghumanized immunoglobulins, can also be found, in Goeddel et al., GENEEXPRESSION TECHNOLOGY METHODS IN ENZYMOLOGY Vol. 185 Academic Press(1991), and Borreback, ANTIBODY ENGINEERING, W. H. Freeman (1992).Additional information concerning the generation, design and expressionof recombinant antibodies can be found in Mayforth, DESIGNINGANTIBODIES, Academic Press, San Diego (1993).

An exemplary process for the production of the recombinant humanizedantibodies of the invention may comprise the following: (A)constructing, by conventional molecular biology methods, an expressionvector comprising an operon that encodes an antibody heavy chain inwhich the CDRs and a minimal portion of the variable region frameworkthat are required to retain donor antibody binding specificity arederived from a non-human immunoglobulin, such as the murine FcγRIIBmonoclonal antibody, and the remainder of the antibody is derived from ahuman immunoglobulin, thereby producing a vector for the expression of ahumanized antibody heavy chain; (B) constructing, by conventionalmolecular biology methods, an expression vector comprising an operonthat encodes an antibody light chain in which the CDRs and a minimalportion of the variable region framework that are required to retaindonor antibody binding specificity are derived from a non-humanimmunoglobulin, such as the murine FcγRIIB monoclonal antibody, and theremainder of the antibody is derived from a human immunoglobulin,thereby producing a vector for the expression of humanized antibodylight chain; (C) transferring the expression vectors to a host cell byconventional molecular biology methods to produce a transfected hostcell for the expression of humanized anti-FcγRIIB antibodies; and (D)culturing the transfected cell by conventional cell culture techniquesso as to produce humanized anti-FcγRIIB antibodies. Host cells may becotransfected with two expression vectors of the invention, the firstvector containing an operon encoding a heavy chain derived polypeptideand the second containing an operon encoding a light chain derivedpolypeptide. The two vectors may contain different selectable markersbut, with the exception of the heavy and light chain coding sequences,are preferably identical. This procedure provides for equal expressionof heavy and light chain polypeptides. Alternatively, a single vectormay be used which encodes both heavy and light chain polypeptides. Thecoding sequences for the heavy and light chains may comprise cDNA orgenomic DNA or both. The host cell used to express the recombinantantibody of the invention may be either a bacterial cell such asEscherichia coli, or preferably a eukaryotic cell. Preferably, amammalian cell such as a chinese hamster ovary cell or HEK-293 cells,may be used. The choice of expression vector is dependent upon thechoice of host cell, and may be selected so as to have the desiredexpression and regulatory characteristics in the selected host cell.Other cell lines that may be used include, but are not limited to,CHO-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).

In a specific embodiment the method for producing a humanized FcγRIIB2B6 antibody comprises the following: RNA from hybridoma cells of 2B6 isconverted to cDNA and the VH and VL segments are PCR amplified using,for example, the RLM-RACE kit (Ambion, Inc.). Gene specific primers forthe VH are used. Examples of such primers for VH include: SJ15R, SEQ IDNO:47 (5′ GGT CAC TGT CAC TGG CTC AGG G 3′) and SJ16R, SEQ ID NO:48 (5′AGG CGG ATC CAG GGG CCA GTG GAT AGA C 3′), and for VL include SJ17R, SEQID NO:49 (5′ GCA CAC GAC TGA GGC ACC TCC AGA TG 3′) and SJ18R, SEQ IDNO:50 (5′ CGG CGG ATC CGA TGG ATA CAG TTG GTG CAG CAT C 3′). The RACEproduct is inserted into a plasmid, e.g., pCR2.1-TOPO using a TOPO TACloning kit (Invitrogen, Inc.). The resulting plasmids are thensubjected to DNA sequencing to determine the VH and VL sequences for2B6. The resulting sequences are translated and the predicted amino acidsequence determined for each. From these sequences the framework (FR)and complementarity determining (CDR) regions are identified as definedby Kabat. The mouse VH is then joined to a human C-Gamma1 constantregion and an Ig leader sequence and inserted into pCI-neo for mammalianexpression. The mouse VL is joined to a human C-kappa segment and an Igleader sequence and also cloned into pCI-neo for mammalian expression.The humanized 2B6 VH consists of the FR segments from the human germlineVH segment VH1-18 and JH6, and the CDR regions of the 2B6 VH. Thehumanized 2B6 VL consists of the FR segments of the human germline VLsegment VK-A26 and JK4, and the CDR regions of 2B6 VL. The humanized VHand VL segments are assembled de novo from oligonucleotides combined andamplified by PCR. The resulting fragment is then combined by PCR with aleader sequence and the appropriate constant region segment cloned intothe expression vector pCI-neo. The DNA sequence of the resultingplasmids is confirmed by sequence analysis. After this procedure lightchain segments having predicted humanized 2B6 VL sequence areidentified. Representative plasmids, pMGx608 (containing a humanized 2B6heavy chain) and pMGx611 (containing a humanized 2B6 light chain withN₅₀→Y and V₅₁→A in CDR2), having ATCC Accession numbers PTA-5963 andPTA-5964, respectively, were deposited under the provisions of theBudapest Treaty with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 7, 2004,respectively, and are incorporated herein by reference.

The general methods for construction of the vectors of the invention,transfection of cells to produce the host cell of the invention, cultureof cells to produce the antibody of the invention are all conventionalmolecular biology methods. Likewise, once produced, the recombinanthumanized antibodies of the invention may be purified by standardprocedures of the art, including cross-flow filtration, ammoniumsulphate precipitation, affinity column chromatography, gelelectrophoresis and the like.

The humanized FcγRIIB specific antibodies of the present invention maybe used in conjunction with, or attached to, other antibodies (or partsthereof) such as human or humanized monoclonal antibodies. These otherantibodies may be reactive with other markers (epitopes) characteristicfor the disease against which the antibodies of the invention aredirected or may have different specificities chosen, for example, torecruit molecules or cells of the human immune system to the diseasedcells. The antibodies of the invention (or parts thereof) may beadministered with such antibodies (or parts thereof) as separatelyadministered compositions or as a single composition with the two agentslinked by conventional chemical or by molecular biological methods.Additionally the diagnostic and therapeutic value of the antibodies ofthe invention may be augmented by labelling the humanized antibodieswith labels that produce a detectable signal (either in vitro or invivo) or with a label having a therapeutic property. Some labels, e.g.,radionucleotides, may produce a detectable signal and have a therapeuticproperty. Examples of radionuclide labels include, but are not limitedto, ¹²⁵I, ¹³¹I, and ¹⁴C. Examples of other detectable labels include afluorescent chromophore such as fluorescein, phycobiliprotein ortetraethyl rhodamine for fluorescence microscopy, an enzyme whichproduces a fluorescent or colored product for detection by fluorescence,absorbance, visible color or agglutination, which produces an electrondense product for demonstration by electron microscopy; or an electrondense molecule such as ferritin, peroxidase or gold beads for direct orindirect electron microscopic visualization. Labels having therapeuticproperties include drugs for the treatment of cancer, such asmethotrexate and the like.

The subject invention provide numerous humanized antibodies specific forthe FcγRIIB based on the discovery that the CDR regions of the murinemonoclonal antibody could be spliced into a human acceptor framework soas to produce a humanized recombinant antibody specific for the FcγRIIB.Preferred humanized FcγRIIB specific antibodies contain an additionalchange in the framework region (or in other regions) to increasingbinding for FcγRIIB. Particularly preferred embodiments of the inventionare the exemplified humanized antibody molecules that have superiorbinding properties for FcγRIIB.

The invention encompasses standard recombinant DNA methods for preparingDNA sequences which code for the CDR-grafted antibodies of theinvention. DNA sequences may be synthesized completely or in part usingoligonucleotide synthesis techniques. Methods for oligonucleotidedirected synthesis are well known in the art. The invention furtherencompasses site-directed mutagenesis methods such as those known in theart.

Any suitable host cell/vector system may be used for expression of theDNA sequences coding for the CDR-grafted heavy and light chains.Bacterial, e.g., E. coli, and other microbial systems may be used, inparticular for expression of antibody fragments such as Fab and (Fab′)2fragments, and especially FV fragments and single chain antibodyfragments, e.g., single chain FVs. Eucaryotic systems, e.g., mammalianhost cell expression systems, may be used for production of largerCDR-grafted antibody products, including complete antibody molecules.Suitable mammalian host cells include CHO cells and myeloma or hybridomacell lines. Other cell lines that may be used include, but are notlimited to, CHO-K1, NSO, and PER.C6 (Crucell, Leiden, Netherlands).

The donor murine antibodies of the invention may be produced using anymethod known in the art, including those disclosed in U.S. ProvisionalApplication No. 60/403,366, filed on Aug. 14, 2002 and U.S. PatentApplication Publication No. 2004/0185045; both of which are incorporatedherein by reference in their entireties.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CH1 region and at least a portion of the hingeregion of the heavy chain.

For example, antibodies can also be generated using various phagedisplay methods known in the art. In phage display methods, functionalantibody domains are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In a particularembodiment, such phage can be utilized to display antigen bindingdomains, such as Fab and Fv or disulfide-bond stabilized Fv, expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phage,including fd and M13. The antigen binding domains are expressed as arecombinantly fused protein to either the phage gene III or gene VIIIprotein. Examples of phage display methods that can be used to make theimmunoglobulins, or fragments thereof, of the present invention includethose disclosed in Brinkman et al. (1995) “Phage Display OfDisulfide-Stabilized Fv Fragments,” J. Immunol. Methods, 182:41-50; Ameset al. (1995) “Conversion Of Murine Fabs Isolated From A CombinatorialPhage Display Library To Full Length Immunoglobulins,” J. Immunol.Methods, 184:177-186; Kettleborough et al. (1994) “Isolation Of TumorCell-Specific Single-Chain Fv From Immunized Mice Using Phage-AntibodyLibraries And The Re-Construction Of Whole Antibodies From TheseAntibody Fragments,” Eur. J. Immunol., 24:952-958; Persic et al. (1997)“An Integrated Vector System For The Eukaryotic Expression Of AntibodiesOr Their Fragments After Selection From Phage Display Libraries,” Gene,187:9-18; Burton et al. (1994) “Human Antibodies From CombinatorialLibraries,” Advances in Immunology, 57:191-280; PCT Application No.PCT/GB91/01134; PCT Publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′ and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT Publication WO92/22324; Mullinax et al. (1992) “Expression of a Heterodimeric FabAntibody Protein in One Cloning Step,” BioTechniques, 12(6):864-869; andSawai et al. (1995) “Direct Production Of The Fab Fragment Derived FromThe Sperm Immobilizing Antibody Using Polymerase Chain Reaction And cDNAExpression Vectors,” AJRI, 34:26-34; and Better et al. (1988)“Escherichia coli Secretion Of An Active Chimeric Antibody Fragment,”Science, 240:1041-1043 (each of which is incorporated by reference inits entirety). Examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al. (1991) “Protein EngineeringOf Single-Chain Fv Analogs And Fusion Proteins,” Methods in Enzymology,203:46-88; Shu et al. (1993) “Secretion Of A Single-Gene-EncodedImmunoglobulin From Myeloma Cells,” Proc. Natl. Acad. Sci. USA,90:7995-7999; and Skerra et al. (1988) “Assembly Of A FunctionalImmunoglobulin Fv Fragment In Escherichia coli,” Science, 240:1038-1040.

Phage display technology can be used to increase the affinity of anantibody of the invention for FcγRIIB. This technique would be useful inobtaining high affinity antibodies that could be used in thecombinatorial methods of the invention. This technology, referred to asaffinity maturation, employs mutagenesis or CDR walking and re-selectionusing FcγRIIB or an antigenic fragment thereof to identify antibodiesthat bind with higher affinity to the antigen when compared with theinitial or parental antibody (See, e.g., Glaser et al. (1992) “AntibodyEngineering By Codon-Based Mutagenesis In A Filamentous Phage VectorSystem,” J. Immunology 149:3903-3913). Mutagenizing entire codons ratherthan single nucleotides results in a semi-randomized repertoire of aminoacid mutations. Libraries can be constructed consisting of a pool ofvariant clones each of which differs by a single amino acid alterationin a single CDR and which contain variants representing each possibleamino acid substitution for each CDR residue. Mutants with increasedbinding affinity for the antigen can be screened by contacting theimmobilized mutants with labeled antigen. Any screening method known inthe art can be used to identify mutant antibodies with increased avidityto the antigen (e.g., ELISA) (See Wu et al. (1998) “Stepwise In VitroAffinity Maturation Of Vitaxin, An αvβ3-Specific Humanized mAb,” ProcNatl. Acad. Sci. USA 95:6037-6042; Yelton et al. (1995) “AffinityMaturation Of The BR96 Anti-Carcinoma Antibody By Codon-BasedMutagenesis,” J. Immunology 155:1994-2004). CDR walking which randomizesthe light chain is also possible (See Schier et al. (1996) “Isolation OfPicomolar Affinity Anti-c-erbB-2 Single-Chain Fv By Molecular EvolutionOf The Complementarity Determining Regions In The Center Of The AntibodyBinding Site,” J. Mol. Bio. 263:551-567).

IV. Screening for Biological Properties

The humanized antibodies of the invention may be characterized forspecific binding to FcγRIIB using any immunological or biochemical basedmethod known in the art for characterizing, including quantitating theinteraction of the antibody to FcγRIIB. Specific binding of a humanizedantibody of the invention to FcγRIIB may be determined, for example,using immunological or biochemical based methods including, but notlimited to, an ELISA assay, surface plasmon resonance assays,immunoprecipitation assay, affinity chromatography, fluorescenceactivated cell sorting (FACS), and equilibrium dialysis. Immunoassayswhich can be used to analyze immunospecific binding and cross-reactivityof the antibodies of the invention include, but are not limited to,competitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew. Such assays are routine and well known in the art (see, e.g.,Ausubel et al., eds, 1994, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Vol.1, John Wiley & Sons, Inc., New York, which is incorporated by referenceherein in its entirety).

Humanized antibodies of the invention may be characterized for bindingto FcγRIIB using an in vitro ELISA assay. An exemplary ELISA assay foruse in the methods of the invention may comprise the following: 2.5ng/well of soluble FcγRIIb-Fc fusion protein which is prepared inaccordance with methods disclosed in U.S. Provisional Application No.60/439,709 and U.S. application Ser. No. 10/756,153, published as U.S.Patent Application Publication 2004/0265321 A1, both of which areincorporated herein by reference in its entirety, is captured on 96-wellMaxisorp plates by mouse anti-FcγRIIb antibody 3H7 at room temperaturefor 1 hour. A serial of two-fold dilution of conditioned medium of ch2B6or hu2B6Hc/Ch2B6Lc starting from 25 ng/well is added to the each well.The plate is incubated at room temperature for 1 hour, then binding isdetected by HRP conjugated F(ab′)₂ goat anti human IgG F(ab)′2 specificsecondary antibody. After incubation with the secondary antibody forapproximately 45 minutes, the plate is developed using a TMB substrate.After 5 minutes incubation, the reaction is stopped by 1% H₂SO₄. The OD450 nm is read by SOFTmax program. Between each step, the plates arewashed 3 times with PBS/0.1% Tween 20. Plates are blocked by 0.5% BSA inPBS/0.1% Tween 20 for 30 mins at room temperature before adding solubleFcγRIIb-Fc.

Humanized antibodies of the invention may be characterized for bindingto FcγRIIB expressing cells, such as Daudi cells and Rajii cells usingfluorescence activated cell sorting (FACS), using any of the techniquesknown to those skilled in the art. Flow sorters are capable of rapidlyexamining a large number of individual cells (e.g., 10-100 million cellsper hour) (Shapiro et al., PRACTICAL FLOW CYTOMETRY, 1995). Flowcytometers for sorting and examining biological cells are well known inthe art. Known flow cytometers are described, for example, in U.S. Pat.Nos. 4,347,935; 5,464,581; 5,483,469; 5,602,039; 5,643,796; and6,211,477; the entire contents of which are incorporated by referenceherein. Other known flow cytometers are the FACS Vantage™ systemmanufactured by Becton Dickinson and Company, and the COPAS™ systemmanufactured by Union Biometrica. An exemplary FACS analysis forcharacterizing the humanized antibodies of the invention may comprisethe following: Approximately 10⁶ FcγRIIB expressing cells, e.g., Daudicells and Rajii cells, are washed at least once with a buffer such asPBS. Primary antibodies (e.g., Ch2B6, Hu2B6Hc/ch2B6Lc, human IgG1) arediluted, e.g., into 0.5, 0.1, 0.02 μg/mL in PBS/1% BSA and 100 μl ofdiluted antibodies are transferred to the cells. After 30 minsincubation at 4° C., cells are washed once with 1 ml PBS/1% BSA. PEconjugated F(ab′)₂ fragment of goat anti human IgG Fc specific (JacksonImmunoReseach, Inc.) is used as secondary antibody at 1:1000 dilution.After 30 mins incubation at 4° C., the cells are washed once with 1 mlPBS/1% BSA. Then the cells are resuspended in 500 μl of PBS/1% BSA andsubjected to FACS analysis. Other cell lines that may be used in themethods of the invention include, but are not limited to, CHO-K1(hamster cell line) cells transfected with CD32B; CHO-K1 (hamster cellline) cells transfected with CD32A; 293H (human epithelial cell line)cells transfected with CD32B; 293H (human epithelial cell line) cellstransfected with CD32A; Raji (human Burkitt's lymphoma cell line) cells;Daudi (human Burkitt's lymphoma cell line) cells [Raji and Daudi B celllines express only endogenous CD32B]; THP-1 (human monocytic cell line)cells expressing only endogenous CD32A; U937 (human monocytic cell line)cells expressing endogenous CD32A and CD32B; K526; HL60.

Humanized antibodies of the invention may be further characterized byepitope mapping, so that antibodies may be selected that have thegreatest specificity for FcγRIIB compared to FcγRIIA. Epitope mappingmethods of antibodies are well known in the art and encompassed withinthe methods of the invention. In certain embodiments, FcγRIIB, or afusion protein comprising one or more regions of FcγRIIB, may be used inmapping the epitope of an antibody of the invention. In a specificembodiment, the fusion protein contains the amino acid sequence of aregion of an FcγRIIB fused to the Fc portion of human IgG2. Each fusionprotein may further comprise amino acid substitutions and/orreplacements of certain regions of the receptor with the correspondingregion from a homolog receptor, e.g., FcγRIIA, as shown in TABLE 2below. pMGX125 and pMGX132 contain the IgG binding site of the FcγRIIBreceptor, the former with the C-terminus of FcγRIIB and the latter withthe C-terminus of FcγRIIA and can be used to differentiate C-terminusbinding. The others have FcγRIIA substitutions in the IgG binding siteand either the FcγIIA or FcγIIB N-terminus. These molecules can helpdetermine the part of the receptor molecule where the antibodies bind.

TABLE 2 List of the fusion proteins that may be used to investigate theepitope of the monoclonal anti-FcγRIIB antibodies. Residues 172 to 180belong to the IgG binding site of FcγRIIA and B. The specific aminoacids from A sequence are in bold. SEQ SEQ ID ID Plasmid ReceptorN-terminus 172-180 NO: C-terminus NO: pMGX125 RIIB IIB KKFSRSDPN 51APS------SS (IIB) 57 pMGX126 RIIA/B IIA QKFSRLDPN 52 APS------SS (IIB)57 pMGX127 IIA QKFSRLDPT 53 APS------SS (IIB) 57 pMGX128 IIB KKFSRLDPT54 APS------SS (IIB) 57 pMGX129 IIA QKFSHLDPT 55 APS------SS (IIB) 57pMGX130 IIB KKFSHLDPT 56 APS------SS (IIB) 57 pMGX131 IIA QKFSRLDPN 52VPSMGSSS (IIA) 58 pMGX132 IIB KKFSRSDPN 51 VPSMGSSS (IIA) 58 pMGX133RIIA- IIA QKFSRLDPT 53 VPSMGSSS (IIA) 58 131R pMGX134 RIIA- IIAQKFSHLDPT 55 VPSMGSSS (IIA) 58 131H pMGX135 IIB KKFSRLDPT 54 VPSMGSSS(IIA) 58 pMGX136 IIB KKFSHLDPT 56 VPSMGSSS (IIA) 58

Humanized antibodies of the invention may also be assayed using anysurface plasmon resonance based assays known in the art forcharacterizing the kinetic parameters of the interaction of the antibodywith FcγRIIB. Any SPR instrument commercially available including, butnot limited to, BIAcore Instruments, available from Biacore AB (Uppsala,Sweden); IAsys instruments available from Affinity Sensors (Franklin,Mass.); IBIS system available from Windsor Scientific Limited (Berks,UK); SPR-CELLIA systems available from Nippon Laser and Electronics Lab(Hokkaido, Japan); and SPR Detector Spreeta available from TexasInstruments (Dallas, Tex.) can be used in the instant invention. For areview of SPR-based technology, see Mullet et al. (2000) “SurfacePlasmon Resonance-Based Immunoassays,” Methods 22: 77-91; Dong et al.(2002) “Some New Aspects In Biosensors,” Review in Mol. Biotech., 82:303-323; Fivash et al. (1998) “BIAcore For Macromolecular Interaction,”Current Opinion in Biotechnology 9: 97-101; Rich et al. (2000) “AdvancesIn Surface Plasmon Resonance Biosensor Analysis,” Current Opinion inBiotechnology 11: 54-61; all of which are incorporated herein byreference in their entirety. Additionally, any of the SPR instrumentsand SPR based methods for measuring protein-protein interactionsdescribed in U.S. Pat. Nos. 6,373,577; 6,289,286; 5,322,798; 5,341,215;and 6,268,125, all of which are incorporated herein by reference intheir entirety, are contemplated in the methods of the invention.

Briefly, SPR based assays involve immobilizing a member of a bindingpair on a surface, and monitoring its interaction with the other memberof the binding pair in solution in real time. SPR is based on measuringthe change in refractive index of the solvent near the surface thatoccurs upon complex formation or dissociation. The surface onto whichthe immobilization occur is the sensor chip, which is at the heart ofthe SPR technology; the sensor chip consists of a glass surface coatedwith a thin layer of gold and forms the basis for a range of specializedsurfaces designed to optimize the binding of a molecule to the surface.A variety of sensor chips are commercially available especially from thecompanies listed supra, all of which may be used in the methods of theinvention. Examples of sensor chips include those available from BIAcoreAB, Inc., e.g., Sensor Chip CM5, SA, NTA, and HPA. A molecule of theinvention may be immobilized onto the surface of a sensor chip using anyof the immobilization methods and chemistries known in the art,including, but not limited to, direct covalent coupling via aminegroups, direct covalent coupling via sulfhydryl groups, biotinattachment to avidin coated surface, aldehyde coupling to carbohydrategroups, and attachment through the histidine tag with NTA chips.

The invention encompasses characterization of the humanized antibodiesproduced by the methods of the invention using certain characterizationassays for identifying the function of the antibodies of the invention,particularly the activity to modulate FcγRIIB signaling. For example,characterization assays of the invention can measure phosphorylation oftyrosine residues in the ITIM motif of FcγRIIB, or measure theinhibition of B cell receptor-generated calcium mobilization. Thecharacterization assays of the invention can be cell-based or cell-freeassays.

It has been well established in the art that in mast cells coaggregationof FcγRIIB with the high affinity IgE receptor, FcεRI, leads toinhibition of antigen-induced degranulation, calcium mobilization, andcytokine production (Metcalfe et al. (1997) “Mast Cells,” Physiol. Rev.77:1033-1079; Long (1999) “Regulation Of Immune Responses ThroughInhibitory Receptors,” Annu Rev. Immunol 17: 875-904). The moleculardetails of this signaling pathway have been recently elucidated (Ott(2002) “Downstream Of Kinase, p62(dok), Is A Mediator Of Fc gamma IIBInhibition Of Fc epsilon RI SIgnaling,” J. Immunol. 162(9):4430-4439).Once coaggregated with FcεRI, FcγRIIB is rapidly phosphorylated ontyrosine in its ITIM motif, and then recruits Src Homology-2 containinginositol-5-phosphatase (SHIP), an SH2 domain—containing inositalpolyphosphate 5-phosphatase, which is in turn phosphorylated andassociates with Shc and p62^(dok) (p62^(dok) is the prototype of afamily of adaptor molecules, which includes signaling domains such as anaminoterminal pleckstrin homology domain (PH domain), a PTB domain, anda carboxy terminal region containing PXXP motifs and numerousphosphorylation sites (Carpino et al. (1997) “p62(dok): A ConstitutivelyTyrosine-Phosphorylated, GAP-Associated Protein In Chronic MyelogenousLeukemia Progenitor Cells,” Cell, 88: 197-204; Yamanshi et al. (1997)“Identification Of The Abl-And rasGAP-Associated 62 kDa Protein As ADocking Protein, Dok,” Cell, 88:205-211).

The invention encompasses characterizing the anti-FcγRIIB humanizedantibodies of the invention in modulating one or more IgE mediatedresponses. Preferably, cells lines co-expressing the high affinityreceptor for IgE and the low affinity receptor for FcγRIIB will be usedin characterizing the anti-FcγRIIB antibodies of the invention inmodulating IgE mediated responses. In a specific embodiment, cells froma rat basophilic leukemia cell line (RBL-H23; Barsumian et al. (1981)“IgE-Induced Histamine Release From Rat Basophilic Leukemia Cell Lines:Isolation Of Releasing And Nonreleasing Clones,” Eur. J. Immunol.11:317-323, which is incorporated herein by reference in its entirety)transfected with full length human FcγRIIB will be used in the methodsof the invention. RBL-2H3 is a well characterized rat cell line that hasbeen used extensively to study the signaling mechanisms followingIgE-mediated cell activation. When expressed in RBL-2H3 cells andcoaggregated with FcεRI, FcγRIIB inhibits FcεRI-induced calciummobilization, degranulation, and cytokine production (Malbec et al.(1998) “Fc epsilon Receptor I-Associated lyn-Dependent PhosphorylationOf Fc gamma Receptor IIB During Negative Regulation Of Mast CellActivation,” J. Immunol. 160:1647-1658; Daeron et al. (1995) “RegulationOf High-Affinity IgE Receptor-Mediated Mast Cell Activation By MurineLow-Affinity IgG Receptors,” J. Clin. Invest. 95:577-585; Ott et al.(2002) “Downstream Of Kinase, p62(dok), Is A Mediator Of Fc gamma IIBInhibition Of Fc epsilon RI Signaling,” J. of Immunol. 168:4430-4439).

In some embodiments, the invention encompasses characterizing theanti-FcγRIIB humanized antibodies of the invention for inhibition ofFcεRI induced mast cell activation. For example, cells from a ratbasophilic leukemia cell line (RBL-H23; Barsumian et al. (1981)“IgE-Induced Histamine Release From Rat Basophilic Leukemia Cell Lines:Isolation Of Releasing And Nonreleasing Clones,” Eur. J. Immunol.11:317-323) that have been transfected with FcγRIIB are sensitized withIgE and stimulated either with F(ab′)₂ fragments of rabbit anti-mouseIgG, to aggregate FcεRI alone, or with whole rabbit anti-mouse IgG tocoaggregate FcγRIIB and FcεRI. In this system, indirect modulation ofdown stream signaling molecules can be assayed upon addition ofantibodies of the invention to the sensitized and stimulated cells. Forexample, tyrosine phosphorylation of FcγRIIB and recruitment andphosphorylation of SHIP, activation of MAP kinase family members,including, but not limited to Erk1, Erk2, JNK, or p38; and tyrosinephosphorylation of p62^(dok) and its association with SHIP and RasGAPcan be assayed.

One exemplary assay for determining the inhibition of FcεRI induced mastcell activation by the antibodies of the invention can comprise of thefollowing: transfecting RBL-H23 cells with human FcγRIIB; sensitizingthe RBL-H23 cells with IgE; stimulating RBL-H23 cells with eitherF(ab′)₂ of rabbit anti-mouse IgG (to aggregate FcεRI alone and elicitFcεRI-mediated signaling, as a control), or stimulating RBL-H23 cellswith whole rabbit anti-mouse IgG to (to coaggregate FcγRIIB and FcεRI,resulting in inhibition of FcεRI-mediated signaling). Cells that havebeen stimulated with whole rabbit anti-mouse IgG antibodies can befurther pre-incubated with the antibodies of the invention. MeasuringFcεRI-dependent activity of cells that have been pre-incubated with theantibodies of the invention and cells that have not been pre-incubatedwith the antibodies of the invention, and comparing levels ofFcεRI-dependent activity in these cells, would indicate a modulation ofFcεRI-dependent activity by the antibodies of the invention.

The exemplary assay described above can be used, for example, toidentify antibodies that block ligand (IgG) binding to FcγRIIB receptorand antagonize FcγRIIB-mediated inhibition of FcεRI signaling bypreventing coaggregating of FcγRIIB and FcεRI. This assay likewiseidentifies antibodies that enhance coaggregation of FcγRIIB and FcεRIand agonize FcγRIIB-mediated inhibition of FcεRI signaling by promotingcoaggregating of FcγRIIB and FcεRI.

In a preferred embodiment, FcεRI-dependent activity is at least one ormore of the following: modulation of downstream signaling molecules,e.g., modulation of phosphorylation state of FcγRIIB, modulation of SHIPrecruitment, modulation of MAP Kinase activity, modulation ofphosphorylation state of SHIP, modulation of SHIP and Shc associationSHIP and Shc, modulation of the phosphorylation state of p62^(dok),modulation of p62^(dok) and SHIP association, modulation of p62^(dok)and RasGAP association, modulation of calcium mobilization, modulationof degranulation, and modulation of cytokine production. In yet anotherpreferred embodiment, FcεRI-dependent activity is serotonin releaseand/or extracellular Ca⁺⁺ influx and/or IgE dependent mast cellactivation. It is known to one skilled in the art that coaggregation ofFcγRIIB and FcεRI stimulates FcγRIIB tyrosine phosphorylation,stimulates recruitment of SHIP, stimulates SHIP tyrosine phosphorylationand association with Shc, and inhibits activation of MAP kinase familymembers including, but not limited to, Erk1, Erk2, JNK, p38. It is alsoknown to those skilled in the art that coaggregation of FcγRIIB andFcεRI stimulates enhanced tyrosine phosphorylation of p62^(dok) and itsassociation with SHIP and RasGAP.

In some embodiments, the anti-FcγRIIB humanized antibodies of theinvention are characterized for their ability to modulate an IgEmediated response by monitoring and/or measuring degranulation of mastcells or basophils, preferably in a cell-based assay. Preferably, mastcells or basophils for use in such assays have been engineered tocontain human FcγRIIB using standard recombinant methods known to oneskilled in the art. In a specific embodiment the anti-FcγRIIB antibodiesof the invention are characterized for their ability to modulate an IgEmediated response in a cell-based β-hexosaminidase (enzyme contained inthe granules) release assay. β-hexosaminidase release from mast cellsand basophils is a primary event in acute allergic and inflammatorycondition (Aketani et al. (2001) “Correlation Between Cytosolic CalciumConcentration And Degranulation In RBL-2H3 Cells In The Presence OfVarious Concentrations Of Antigen-Specific IgEs,” Immunol. Lett. 75:185-189; Aketani et al. (2000) “A Screening Method For Antigen-SpecificIgE Using Mast Cells Based On Intracellular Calcium Signaling,” Anal.Chem. 72: 2653-2658). Release of other inflammatory mediators including,but not limited to, serotonin and histamine may be assayed to measure anIgE mediated response in accordance with the methods of the invention.Although not intending to be bound by a particular mechanism of action,release of granules such as those containing β-hexosaminidase from mastcells and basophils is an intracellular calcium concentration dependentprocess that is initiated by the cross-linking of FcεRIs withmultivalent antigen.

One exemplary assay for characterizing the anti-FcγRIIB humanizedantibodies of the invention in mediating an IgE mediated response is aβ-hexosaminidase release assay comprising the following: transfectingRBL-H23 cells with human FcγRIIB; sensitizing the cells with mouse IgEalone or with mouse IgE and an anti-FcγRIIB antibody of the invention;stimulating the cells with various concentrations of goat anti-mouseF(ab)₂, preferably in a range from 0.03 μg/mL to 30 μg/mL for about 1hour; collecting the supernatant; lysing the cells; and measuring theβ-hexosaminidase activity released in the supernatant by a colorometricassay, e.g., using p-nitrophenyl N-acetyl-β-D-glucosaminide. Thereleased β-hexosaminidase activity is expressed as a percentage of thereleased activity to the total activity. The released β-hexosaminidaseactivity will be measured and compared in cells treated with antigenalone; IgE alone; IgE and an anti-FcγRIIB antibody of the invention.Although not intending to be bound by a particular mechanism of action,once cells are sensitized with mouse IgE alone and challenged withF(ab)₂ fragments of a polyclonal goat anti-mouse IgG, aggregation andcross linking of FcεRI occurs since the polyclonal antibody recognizesthe light chain of the murine IgE bound to the FcεRI, which in turnleads to mast cell activation and degranulation. On the other hand, whencells are sensitized with mouse IgE and an anti-FcγRIIB antibody of theinvention and challenged with F(ab)₂ fragments of a polyclonal goatanti-mouse IgG; cross linking of FcεRI and FcγRIIB occurs, resulting ininhibition of FcεRI induced degranulation. In either case, goat antimouse F(ab)₂ induces a dose-dependent β-hexoaminidase release. In someembodiments, the anti-FcγRIIB antibodies bound to the FcγRIIB receptorand cross linked to FcεRI do not affect the activation of the inhibitorypathway, i.e., there is no alteration in the level of degranulation inthe presence of an anti-FcγRIIB antibody. In other embodiments, theanti-FcγRIIB antibodies mediate a stronger activation of the inhibitoryreceptor, FcγRIIB, when bound by the anti-FcγRIIB antibody, allowingeffective cross linking to FcεRI and activation of the inhibitorypathway of homo-aggregated FcγRIIB.

The invention also encompasses characterizing the effect of theanti-FcγRIIB humanized antibodies of the invention on IgE mediated cellresponse using calcium mobilization assays using methodologies known toone skilled in the art. An exemplary calcium mobilization assay maycomprise the following: priming basophils or mast cells with IgE;incubating the cells with a calcium indicator, e.g., Fura 2; stimulatingcells as described supra; and monitoring and/or quantitatingintracellular calcium concentration for example by using flow cytometry.The invention encompasses monitoring and/or quantitating intracellularcalcium concentration by any method known to one skilled in the art.(See, e.g., Aketani et al. (2001) “Correlation Between Cytosolic CalciumConcentration And Degranulation In RBL-2H3 Cells In The Presence OfVarious Concentrations Of Antigen-Specific IgEs,” Immunol. Lett. 75:185-189; Oka et al. (2002) “FcepsilonRI Cross-Linking-Induced ActinAssembly Mediates Calcium Signalling In RBL-2H3 Mast Cells,” British J.of Pharm 136:837-845; Ott et al. (2002) “Downstream Of Kinase, p62(dok),Is A Mediator Of Fc gamma IIB Inhibition Of Fc epsilon RI Signaling,” J.of Immunol. 168:4430-4439 and Mahmoud et al. (2001) “Microdomains ofHigh Calcium Are Not Required for Exocytosis in RBL-2H3 Mucosal MastCells,” J. of Cell Biol., 153(2):339-349; all of which are incorporatedherein by reference.

In preferred embodiments, anti-FcγRIIB humanized antibodies of theinvention inhibit IgE mediated cell activation. In other embodiments,the anti-FcγRIIB antibodies of the invention block the inhibitorypathways regulated by FcγRIIB or block the ligand binding site onFcγRIIB and thus enhance immune response.

In some embodiments, if human mast cells have a low expression ofendogenous FcγRIIB, as determined using standard methods known in theart, e.g., FACS staining, it may be difficult to monitor and/or detectdifferences in the activation of the inhibitory pathway mediated by theanti-FcγRIIB antibodies of the invention. The invention thus encompassesalternative methods, whereby the FcγRIIB expression may be upregulatedusing cytokines and particular growth conditions. FcγRIIB has beendescribed to be highly up-regulated in human monocyte cell lines, e.g.,THP1 and U937, (Tridandapani et al. (2002) “Regulated Expression AndInhibitory Function Of Fcgamma RIIb In Human Monocytic Cells,” J. Biol.Chem., 277(7):5082-5089) and in primary human monocytes (Pricop et al.(2001) “Differential Modulation Of Stimulatory And Inhibitory Fc GammaReceptors On Human Monocytes By Th1 And Th2 Cytokines,” J. of Immunol.,166: 531-537) by IL4. Differentiation of U937 cells with dibutyrylcyclic AMP has been described to increase expression of FcγRII (Cameronet al. (2002) “Differentiation Of The Human Monocyte Cell Line, U937,With Dibutyryl cyclicAMP Induces The Expression Of The Inhibitory FcReceptor, FcγRIIb,” Immunology Letters 83, 171-179). Thus, theendogenous FcγRIIB expression in human mast cells for use in the methodsof the invention may be up-regulated using cytokines, e.g., IL-4, IL-13,in order to enhance sensitivity of detection.

The invention also encompasses characterizing the humanized anti-FcγRIIBantibodies of the invention for inhibition of B-cell receptor(BCR)-mediated signaling. BCR-mediated signaling can include at leastone or more down stream biological responses, such as activation andproliferation of B cells, antibody production, etc. Coaggregation ofFcγRIIB and BCR leads to inhibition of cell cycle progression andcellular survival. Further, coaggregation of FcγRIIB and BCR leads toinhibition of BCR-mediated signaling.

Specifically, BCR-mediated signaling comprises at least one or more ofthe following: modulation of down stream signaling molecules (e.g.,phosphorylation state of FcγRIIB, SHIP recruitment, localization of Btkand/or PLCγ, MAP kinase activity, recruitment of Akt (anti-apoptoticsignal), calcium mobilization, cell cycle progression, and cellproliferation.

Although numerous effector functions of FcγRIIB-mediated inhibition ofBCR signaling are mediated through SHIP, recently it has beendemonstrated that lipopolysaccharide (LPS)-activated B cells from SHIPdeficient mice exhibit significant FcγRIIB-mediated inhibition ofcalcium mobilization, Ins(1,4,5)P₃ production, and Erk and Aktphosphorylation (Brauweiler et al. (2001) “Partially Distinct MolecularMechanisms Mediate Inhibitory FcgammaRIIB Signaling In Resting AndActivated B Cells,” Journal of Immunology, 167(1):204-211). Accordingly,ex vivo B cells from SHIP deficient mice can be used to characterize theantibodies of the invention. One exemplary assay for determiningFcγRIIB-mediated inhibition of BCR signaling by the antibodies of theinvention can comprise the following: isolating splenic B cells fromSHIP deficient mice, activating said cells with lipopolysaccharide, andstimulating said cells with either F(ab′)₂ anti-IgM to aggregate BCR orwith anti-IgM to coaggregate BCR with FcγRIIB. Cells that have beenstimulated with intact anti-IgM to coaggregate BCR with FcγRIIB can befurther pre-incubated with the antibodies of the invention.FcγRIIB-dependent activity of cells can be measured by standardtechniques known in the art and used for, e.g., comparing the level ofFcγRIIB-dependent activity in cells that have been pre-incubated withthe antibodies of the invention and cells that have not beenpre-incubated, and comparing the levels would indicate a modulation ofFcγRIIB-dependent activity by the antibodies of the invention.

Measuring FcγRIIB-dependent activity can include, for example, measuringintracellular calcium mobilization by flow cytometry, measuringphosphorylation of Akt and/or Erk, measuring BCR-mediated accumulationof PI(3,4,5)P₃, or measuring FcγRIIB-mediated proliferation B cells.

The assays can be used, for example, to identify antibodies thatmodulate FcγRIIB-mediated inhibition of BCR signaling by blocking theligand (IgG) binding site to FcγRIIB receptor and antagonizingFcγRIIB-mediated inhibition of BCR signaling by preventing coaggregationof FcγRIIB and BCR. The assays can also be used to identify antibodiesthat enhance coaggregation of FcγRIIB and BCR and agonizeFcγRIIB-mediated inhibition of BCR signaling.

The invention relates to characterizing the humanized anti-FcγRIIBantibodies of the invention for FcγRII-mediated signaling in humanmonocytes/macrophages. Coaggregation of FcγRIIB with a receptor bearingthe immunoreceptor tyrosine-based activation motif (ITAM) acts todown-regulate FcγR-mediated phagocytosis using SHIP as its effector(Tridandapani et al. (2002) “Regulated Expression And InhibitoryFunction Of Fcgamma RIIb In Human Monocytic Cells,” J. Biol. Chem.,277(7):5082-5089). Coaggregation of FcγRIIA with FcγRIIB results inrapid phosphorylation of the tyrosine residue on FcγRIIB's ITIM motif,leading to an enhancement in phosphorylation of SHIP, association ofSHIP with Shc, and phosphorylation of proteins having the molecularweight of 120 and 60-65 kDa. In addition, coaggregation of FcγRIIA withFcγRIIB results in down-regulation of phosphorylation of Akt, which is aserine-threonine kinase that is involved in cellular regulation andserves to suppress apoptosis.

The invention further encompasses characterizing the humanizedanti-FcγRIIB antibodies of the invention for their inhibition ofFcγR-mediated phagocytosis in human monocytes/macrophages. For example,cells from a human monocytic cell line, THP-1 can be stimulated eitherwith Fab fragments of mouse monoclonal antibody IV.3 against FcγRIIA(Medarex, Inc.) and goat anti-mouse antibody (to aggregate FcγRIIAalone), or with whole IV.3 mouse monoclonal antibody and goat anti-mouseantibody (to coaggregate FcγRIIA and FcγRIIB). In this system,modulation of down stream signaling molecules, such as tyrosinephosphorylation of FcγRIIB, phosphorylation of SHIP, association of SHIPwith Shc, phosphorylation of Akt, and phosphorylation of proteins havingthe molecular weight of 120 and 60-65 kDa can be assayed upon additionof antibodies of the invention to the stimulated cells. In addition,FcγRIIB-dependent phagocytic efficiency of the monocyte cell line can bedirectly measured in the presence and absence of the antibodies of theinvention.

Another exemplary assay for determining inhibition of FcγR-mediatedphagocytosis in human monocytes/macrophages by the antibodies of theinvention can comprise the following: stimulating THP-1 cells witheither Fab of IV.3 mouse anti-FcγRIIA antibody and goat anti-mouseantibody (to aggregate FcγRIIA alone and elicit FcγRIIA-mediatedsignaling); or with mouse anti-FcγRII antibody and goat anti-mouseantibody (to coaggregate FcγRIIA and FcγRIIB and inhibitingFcγRIIA-mediated signaling). Cells that have been stimulated with mouseanti-FcγRII antibody and goat anti-mouse antibody can be furtherpre-incubated with the antibodies of the invention. MeasuringFcγRIIA-dependent activity of stimulated cells that have beenpre-incubated with antibodies of the invention and cells that have notbeen pre-incubated with the antibodies of the invention and comparinglevels of FcγRIIA-dependent activity in these cells would indicate amodulation of FcγRIIA-dependent activity by the antibodies of theinvention.

The exemplary assay described can be used, for example, to identifyantibodies that block ligand binding of FcγRIIB receptor and antagonizeFcγRIIB-mediated inhibition of FcγRIIA signaling by preventingcoaggregation of FcγRIIB and FcγRIIA. This assay likewise identifiesantibodies that enhance coaggregation of FcγRIIB and FcγRIIA and agonizeFcγRIIB-mediated inhibition of FcγRIIA signaling.

In another embodiment of the invention, the invention relates tocharacterizing the function of the humanized antibodies of the inventionby measuring the ability of THP-1 cells to phagocytose fluoresceinatedIgG-opsonized sheep red blood cells (SRBC) by methods previouslydescribed (Tridandapani et al. (2002) “Regulated Expression AndInhibitory Function Of Fcgamma RIIb In Human Monocytic Cells,” J. Biol.Chem., 277(7):5082-5089). For example, an exemplary assay for measuringphagocytosis comprises: treating THP-1 cells with the antibodies of theinvention or with a control antibody that does not bind to FcγRII,comparing the activity levels of said cells, wherein a difference in theactivities of the cells (e.g., rosetting activity (the number of THP-1cells binding IgG-coated SRBC), adherence activity (the total number ofSRBC bound to THP-1 cells), and phagocytic rate) would indicate amodulation of FcγRIIA-dependent activity by the antibodies of theinvention. This assay can be used to identify, for example, antibodiesthat block ligand binding of FcγRIIB receptor and antagonizeFcγRIIB-mediated inhibition of phagocytosis. This assay can alsoidentify antibodies that enhance FcγRIIB-mediated inhibition of FcγRIIAsignaling.

In a preferred embodiment, the humanized antibodies of the inventionmodulate FcγRIIB-dependent activity in human monocytes/macrophages in atleast one or more of the following ways: modulation of downstreamsignaling molecules (e.g., modulation of phosphorylation state ofFcγRIIB, modulation of SHIP phosphorylation, modulation of SHIP and Shcassociation, modulation of phosphorylation of Akt, modulation ofphosphorylation of additional proteins around 120 and 60-65 kDa) andmodulation of phagocytosis.

The invention encompasses characterization of the humanized antibodiesof the invention using assays known to those skilled in the art foridentifying the effect of the antibodies on effector cell function oftherapeutic antibodies, e.g., their ability to enhance tumor-specificADCC activity of therapeutic antibodies. Therapeutic antibodies that maybe used in accordance with the methods of the invention include, but arenot limited to, anti-tumor antibodies, anti-viral antibodies,anti-microbial antibodies (e.g., bacterial and unicellular parasites),examples of which are disclosed herein. In particular, the inventionencompasses characterizing the antibodies of the invention for theireffect on FcγR-mediated effector cell function of therapeuticantibodies, e.g., tumor-specific monoclonal antibodies. Examples ofeffector cell functions that can be assayed in accordance with theinvention, include, but are not limited to, antibody-dependent cellmediated cytotoxicity, phagocytosis, opsonization, opsonophagocytosis,C1q binding, and complement dependent cell mediated cytotoxicity. Anycell-based or cell free assay known to those skilled in the art fordetermining effector cell function activity can be used (for effectorcell assays, see Perussia et al. (2000) “Assays For Antibody-DependentCell-Mediated Cytotoxicity (ADCC) And Reverse ADCC (RedirectedCytotoxicity) In Human Natural Killer Cells,” Methods Mol. Biol. 121:179-192; Baggiolini et al. (1998) “Cellular Models For The Detection AndEvaluation Of Drugs That Modulate Human Phagocyte Activity,”Experientia, 44(10):841-848; Lehmann et al. (2000) “Phagocytosis:Measurement By Flow Cytometry,” J. Immunol. Methods, 243:229-242; Brown(1994) “Chapter 8: In Vitro Assays of Phagocytic Function of HumanPeripheral Blood Leukocytes: Receptor Modulation and SignalTransduction,” Methods Cell Biol., 45: 147-164; Munn et al. (1990)“Phagocytosis Of Tumor Cells By Human Monocytes Cultured In RecombinantMacrophage Colony-Stimulating Factor,” J. Exp. Med., 172:231-237,Abdul-Majid et al. (2002) “Fc Receptors Are Critical For AutoimmuneInflammatory Damage To The Central Nervous System In ExperimentalAutoimmune Encephalomyelitis,” Scand. J. Immunol. 55:70-81; Ding et al.(1998) “Two Human T Cell Receptors Bind in a Similar Diagonal Mode tothe HLA-A2/Tax Peptide Complex Using Different TCR Amino Acids,”Immunity 8:403-411, each of which is incorporated by reference herein inits entirety).

Antibodies of the invention can be assayed for their effect onFcγR-mediated ADCC activity of therapeutic antibodies in effector cells,e.g., natural killer cells, using any of the standard methods known tothose skilled in the art (see e.g., Perussia et al. (2000) “Assays ForAntibody-Dependent Cell-Mediated Cytotoxicity (ADCC) And Reverse ADCC(Redirected Cytotoxicity) In Human Natural Killer Cells,” Methods Mol.Biol. 121: 179-192). “Antibody-dependent cell-mediated cytotoxicity” and“ADCC”, as used herein, carry their ordinary and customary meaning inthe art and refer to an in vitro cell-mediated reaction in whichnonspecific cytotoxic cells that express FcγRs (e.g., monocytic cellssuch as Natural Killer (NK) cells and macrophages) recognize boundantibody on a target cell and subsequently cause lysis of the targetcell. In principle, any effector cell with an activating FcγR can betriggered to mediate ADCC. The primary cells for mediating ADCC are NKcells which express only FcγRIII, whereas monocytes, depending on theirstate of activation, localization, or differentiation, can expressFcγRI, FcγRII, and FcγRIII. For a review of FcγR expression onhematopoietic cells, see, e.g., Ravetch et al. (1991) “Fc Receptors,”Annu. Rev. Immunol., 9:457-492, which is incorporated herein byreference in its entirety.

Effector cells are leukocytes which express one or more FcγRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Effector cells that may beused in the methods of the invention include, but are not limited to,peripheral blood mononuclear cells (PBMC), natural killer (NK) cells,monocytes, and neutrophils; with PBMCs and NK cells being preferred. Theeffector cells may be isolated from a native source thereof, e.g., fromblood or PBMCs as described herein. Preferably, the effector cells usedin the ADCC assays of the invention are peripheral blood mononuclearcells (PBMC) that are preferably purified from normal human blood, usingstandard methods known to one skilled in the art, e.g., usingFicoll-Paque density gradient centrifugation. For example, PBMCs may beisolated by layering whole blood onto Ficoll-Hypaque and spinning thecells at 500 g, at room temperature for 30 minutes. The leukocyte layercan be harvested as effector cells. Other effector cells that may beused in the ADCC assays of the invention include, but are not limitedto, monocyte-derived macrophages (MDMs). MDMs that are used as effectorcells in the methods of the invention are preferably obtained as frozenstocks or used fresh (e.g., from Advanced Biotechnologies, MD). In mostpreferred embodiments, elutriated human monocytes are used as effectorcells in the methods of the invention. Elutriated human monocytesexpress activating receptors, FcγRIIIA and FcγRIIA and the inhibitoryreceptor, FcγRIIB. Human monocytes are commercially available and may beobtained as frozen stocks, thawed in basal medium containing 10% humanAB serum or in basal medium with human serum containing cytokines.Levels of expression of FcγRs in the cells may be directly determined;e.g. using FACS analysis. Alternatively, cells may also be allowed tomature to macrophages in culture. The level of FcγRIIB expression may beincreased in macrophages. Antibodies that may be used in determining theexpression level of FcγRs include but are not limited to anti-humanFcγRIIA antibodies, e.g., IV.3-FITC; anti-FcγRI antibodies, e.g., 32.2FITC; and anti-FcγRIIIA antibodies, e.g., 3G8-PE.

Target cells used in the ADCC assays of the invention include, but arenot limited to, breast cancer cell lines, e.g., SK-BR-3 with ATCCaccession number HTB-30 (see, e.g., Trempe et al. (1976) “Human BreastCancer In Culture,” Recent Results in Cancer Res. 57:33-41);B-lymphocytes; cells derived from Burkitts lymphoma, e.g., Raji cellswith ATCC accession number CCL-86 (see, e.g., Epstein et al. (1965)“Characteristics And Mode Of Growth Of Tissue Culture Strain (EB1) OfHuman Lymphoblasts From Burkitt's Lymphoma,” J. Natl. Cancer Inst. 34:231-240), Daudi cells with ATCC accession number CCL-213 (see, e.g.,Klein et al. (1968) “Surface IgM-kappa Specificity On A Burkitt LymphomaCell In Vivo And In Derived Culture Lines,” Cancer Res. 28:1300-1310);ovarian carcinoma cell lines, e.g., OVCAR-3 with ATCC accession numberHTB-161 (see, e.g., Hamilton et al. (1983) “Characterization Of A HumanOvarian Carcinoma Cell Line (NIH:OVCAR-3) With Androgen And EstrogenReceptors,” Cancer Res. 43:5379-5389), SK-OV-3, PA-1, CAOV3, OV-90, andIGROV-1 (available from the NCI repository; Benard et al. (1985)“Characterization of a Human Ovarian Adenocarcinoma Line, IGROV1, inTissue Culture and in Nude Mice,” Cancer Research, 45:4970-4979; whichis incorporated herein by reference in its entirety). The target cellsmust be recognized by the antigen binding site of the antibody to beassayed. The target cells for use in the methods of the invention mayhave low, medium, or high expression level of a cancer antigen. Theexpression levels of the cancer antigen may be determined using commonmethods known to one skilled in the art, e.g., FACS analysis. Forexample, the invention encompasses the use of ovarian cancer cells suchas IGROV-1, wherein Her2/neu is expressed at different levels, orOV-CAR-3 (ATCC Accession Number HTB-161; characterized by a lowerexpression of Her2/neu than SK-BR-3, the breast carcinoma cell line).Other ovarian carcinoma cell lines that may be used as target cells inthe methods of the invention include OVCAR-8 (Hamilton et al. (1983)“Characterization Of A Human Ovarian Carcinoma Cell Line (NIH:OVCAR-3)With Androgen And Estrogen Receptors,” Cancer Res. 43:5379-5389, whichis incorporated herein by reference in its entirety); SK-OV-3 (ATCCAccession Number HTB-77); Caov-3 (ATCC Accession Number HTB-75); PA-1(ATCC Accession Number CRL-1572); OV-90 (ATCC Accession NumberCRL-11732); and OVCAR-4. Other breast cancer cell lines that may be usedin the methods of the invention include BT-549 (ATCC Accession NumberHTB-122), MCF7 (ATCC Accession Number HTB-22), and Hs578T (ATCCAccession Number HTB-126), all of which are available from the NCIrepository and ATCC. Other cell lines that may be used in the methods ofthe invention include, but are not limited to, CCRF-CEM (leukemia);HL-60 (TB, leukemia); MOLT-4 (leukemia); RPMI-8226 (leukemia); SR(leukemia); A549 (Non-small cell lung); EKVX (Non-small cell lung);HOP-62 (Non-small cell lung); HOP-92 (Non-small cell lung); NC1-H226(Non-small cell lung); NC1-H23 (Non-small cell lung); NC1-H322M(Non-small cell lung); NC1-H460 (Non-small cell lung); NC1-H522(Non-small cell lung); COLO 205 (Colon); HCC-2998 (Colon); HCT-116(Colon); HCT-15 (Colon); HT29 (Colon); KM12 (Colon); SW-620 (Colon);SF-268 (CNS); SF-295 (CNS); SF-539 (CNS); SNB-19 (CNS); SNB-75 (CNS);U251 (CNS); LOX 1MV1 (Melanoma); MALME-3M (Melanoma); M14 (Melanoma);SK-MEL-2 (Melanoma); SK-MEL-28 (Melanoma); SK-MEL-5 (Melanoma); UACC-257(Melanoma); UACC-62 (Melanoma); IGR-OV1 (Ovarian); OVCAR-3, 4, 5, 8(Ovarian); SK-OV-3 (Ovarian); 786-0 (Renal); A498 (Renal); ACHN (Renal);CAKl-1 (Renal); SN12C(Renal); TK-10 (Renal); UO-31 (Renal); PC-3C(Prostate); DU-145 (Prostate); NCl/ADR-RES (Breast); MDA-MB-231/ATCC(Breast); MDA-MB-435 (Breast); DMS 114 (Small-cell lung); and SHP-77(Small-cell lung); all of which are available from the NCl.

An exemplary assay for determining the effect of the antibodies of theinvention on the ADCC activity of therapeutic antibodies is based on a⁵¹Cr release assay comprising: labeling target cells with [⁵¹Cr]Na₂CrO₄(this cell-membrane permeable molecule is commonly used for labelingsince it binds cytoplasmic proteins and although spontaneously releasedfrom the cells with slow kinetics, it is released massively followingtarget cell lysis); preferably, the target cells express one or moretumor antigens, osponizing the target cells with one or more antibodiesthat immunospecifically bind the tumor antigens expressed on the cellsurface of the target cells, in the presence and absence of an antibodyof the invention, e.g., 2B6, 3H7, combining the opsonized radiolabeledtarget cells with effector cells in a microtitre plate at an appropriateratio of target cells to effector cells; incubating the mixture of cellspreferably for 16-18 hours, preferably at 37° C.; collectingsupernatants; and analyzing the radioactivity in the supernatantsamples. The cytotoxicity of the therapeutic antibodies in the presenceand absence of the antibodies of the invention can then be determined,for example using the following formula: Percent specificlysis=(Experimental lysis-antibody-independent lysis/maximallysis-antibody independent lysis)×100%. A graph can be generated byvarying either the target:effector cell ratio or antibody concentration.

In yet another embodiment, the antibodies of the invention arecharacterized for antibody dependent cellular cytotoxicity (ADCC) inaccordance with the method described earlier; see, e.g., Ding et al.(1998) “Two Human T Cell Receptors Bind in a Similar Diagonal Mode tothe HLA-A2/Tax Peptide Complex Using Different TCR Amino Acids,”Immunity 8:403-411, which is incorporated herein by reference in itsentirety.

In some embodiments, the invention encompasses characterizing thefunction of the antibodies of the invention in enhancing ADCC activityof therapeutic antibodies in an in vitro based assay and/or in an animalmodel.

In a specific embodiment, the invention encompasses determining thefunction of the humanized antibodies of the invention in enhancing tumorspecific ADCC using an ovarian cancer model and/or breast cancer model.

Preferably, the ADCC assays of the invention are done using more thanone cancer cell line, characterized by the expression of at least onecancer antigen, wherein the expression level of the cancer antigen isvaried among the cancer cell lines used. Although not intending to bebound by a particular mechanism of action, performing ADCC assays inmore than one cell line wherein the expression level of the cancerantigen is varied, will allow determination of stringency of tumorclearance of the antibodies of the invention. In one embodiment, theADCC assays of the invention are done using cancer cell lines withdifferent levels of expression of a cancer antigen.

In an exemplary assay, OVCAR3, an ovarian carcinoma cell line, can serveas the tumor target expressing the tumor antigens, Her2/neu and TAG-72;human monocytes that express the activating FcγRIIIA and FcγRIIA andinhibitory FcγRIIB, can be used as effectors; and tumor specific murineantibodies, ch4D5 and chCC49, can be used as the tumor specificantibodies. OVCAR-3 cells are available from ATCC (Accession NumberHTB-161). Preferably, OVCAR-3 cells are propagated in mediumsupplemented with 0.01 mg/ml bovine insulin. 5×10⁶ viable OVCAR-3 cellsmay be injected subcutaneously (s.c) into age and weight matched nudeathymic mice with Matrigel (Becton Dickinson). The estimated weight ofthe tumor can be calculated by the formula: length-(width)²/2, andpreferably does not exceed 3 grams. Anchorage-dependent tumor can beisolated after 6-8 weeks, and the cells can be dissociated by adding 1μg of Collagenase (Sigma) per gram of tumor and a 5 mg/mL RNase, passedthrough a cell strainer and nylon mesh to isolate cells. Cells can thenbe frozen for long-term storage for s.c. injection for establishment ofthe xenograft model.

Hybridomas secreting CC49 and 4D5 antibodies are available with ATCCAccession Numbers HB-9459 and CRL-3D463 and the heavy chain and lightchain nucleotide sequences are in the public domain (see, e.g., Murrayet al. (1994) “Phase II Radioimmunotherapy Trial With 131I-CC49 InColorectal Cancer,” Cancer 73:1057-1066, Yamamoto et al. (1986)“Similarity Of Protein Encoded By The Human c-erb-B-2 Gene To EpidermalGrowth Factor Receptor,” Nature, 319:230-234; both of which areincorporated herein by reference in their entirety). Preferably, the 4D5and CC49 antibodies are chimerized using standard methods known to oneskilled in the art so that the human Fc sequence, e.g., human constantregion of IgG1, is grafted onto the variable region of the murineantibodies in order to provide the effector function. The chimeric 4D5and CC49 antibodies bind via their variable region to the target celllines and via their Fc region to FcγRs expressed on human effectorcells. CC49 is directed to TAG-72, a high molecular weight mucin that ishighly expressed on many adenocarcinoma cells and ovarian carcinoma(Lottich et al. (1985) “Tumor-Associated Antigen TAG-72: Correlation OfExpression In Primary And Metastatic Breast Carcinoma Lesions,” BreastCancer Res. Treat. 6:49-56; Mansi et al. (1989) “Diagnosis Of OvarianCancer With Radiolabelled Monoclonal Antibodies: Our Experience Using131I-B72.3,” Int. J. Rad. Appl. Instrum B. 16(2):127-135; Colcher et al.(1991) “In Vivo And In Vitro Clinical Applications Of MonoclonalAntibodies Against TAG-72,” Int. J. Rad. Appl. Instrum B. 18:395-341;all of which are incorporated herein by reference in their entirety).4D5 is directed to human epidermal growth factor receptor 2 (Carter etal. (1992) “Humanization Of An Anti-p185HER2 Antibody For Human CancerTherapy,” Proc. Natl. Acad. Sci. USA, 89: 4285-4289, which isincorporated herein by reference). Antibodies of the invention can thenbe utilized to investigate the enhancement of ADCC activity of the tumorspecific antibodies, by blocking the inhibitory FcγRIIB. Although notintending to be bound by a particular mechanism of action, uponactivation of effector cells that express at least one activating FcγR,e.g., FcγRIIA, the expression of the inhibitory receptor (FcγRIIB) isenhanced and this limits the clearance of tumors as the ADCC activity ofFcγRIIA is suppressed. However, antibodies of the invention can serve asa blocking antibody, i.e., an antibody that will prevent the inhibitorysignal from being activated and thus, the activation signal, e.g., ADCCactivity, will be sustained for a longer period and may result in potenttumor clearance.

Preferably, the humanized antibodies of the invention for use inenhancement of ADCC activity have been modified to comprise at least oneamino acid modification so that binding of their Fc region to FcγR hasbeen diminished, most preferably abolished. In some embodiments, theantibodies of the invention have been modified to comprise at least oneamino acid modification which reduces the binding of the constant domainto an activating FcγR, e.g., FcγRIIIA, FcγRIIA, as compared to a wildtype antibody of the invention while retaining maximal FcγRIIB blockingactivity. Antibodies of the invention may be modified in accordance withany method known to one skilled in the art or disclosed herein. Anyamino acid modification which is known to disrupt effector function maybe used in accordance with the methods of the invention such as thosedisclosed in U.S. Application Ser. Nos. 60/439,498 (filed Jan. 9, 2003);and 60/456,041 (filed Mar. 19, 2003); both of which are incorporatedherein by reference in their entireties. In some embodiments, antibodiesof the invention are modified so that position 265 is modified, e.g.,position 265 is substituted with alanine. In preferred embodiments, themurine constant region of an antibody of the invention is swapped withthe corresponding human constant region comprising a substitution of theamino acid at position 265 with alanine, so that the effector functionis abolished while FcγRIIB blocking activity is maintained. A singleamino acid change at position 265 of IgG1 heavy chain has been shown tosignificantly reduce binding to FcγR based on ELISA assays, and hasresulted in tumor mass reduction (Jefferis et al. (1995) “RecognitionSites On Human IgG For Fc gamma Receptors: The Role Of Glycosylation,”Immunology Letters, 44: 111-117, which is incorporated herein byreference in its entirety). In other embodiments, antibodies of theinvention are modified so that position 297 is modified, e.g., position297 is substituted with glutamine, so that the N-linked glycosylationsite is eliminated (see, e.g., Jefferis et al. (1995) “Recognition SitesOn Human IgG For Fc gamma Receptors: The Role Of Glycosylation,”Immunology Letters, 44: 111-117; Lund et al. (1996) “MultipleInteractions Of IgG With Its Core Oligosaccharide Can ModulateRecognition By Complement And Human Fc Gamma Receptor I And InfluenceThe Synthesis Of Its Oligosaccharide Chains,” J. Immunol.,157:4963-4969; Wright et al, (1994) “Effect Of Altered CH2-AssociatedCarbohydrate Structure On The Functional Properties And In Vivo Fate OfChimeric Mouse-Human Immunoglobulin G1,” J. Exp. Med. 180:1087-1096;White et al. (1997) “Ig N-Glycan Orientation Can Influence InteractionsWith The Complement System,” J. Immunol. 158:426-435; all of which areincorporated herein by reference in their entireties. Modification atthis site has been reported to abolish all interaction with FcγRs. Inpreferred embodiments, the murine constant region of an antibody of theinvention is swapped with the corresponding human constant regioncomprising a substitution of the amino acid at position 265 and/or 297,so that the effector function is abolished while FcγRIIB blockingactivity is maintained.

An exemplary assay for determining the ADCC activity of the tumorspecific antibodies in the presence and absence of the antibodies of theinvention is a non-radioactive europium based fluorescent assay (BATDA,Perkin Elmer) and may comprise the following: labeling the targets cellswith an acteoxylmethyl ester of fluorescence-enhancing ester that formsa hydrophilic ligand (TDA) with the membrane of cells by hydrolysis ofthe esters (this complex is unable to leave the cell and is releasedonly upon lysis of the cell by the effectors); adding the labeledtargets to the effector cells in presence of anti-tumor antibodies andan antibody of the invention; and incubating the mixture of the targetand effector cells for 6 to 16 hours, preferably at 37° C. The extent ofADCC activity can be assayed by measuring the amount of ligand that isreleased and interacts with europium (DELFIA® reagent; PerkinElmer). Theligand and the europium form a very stable and highly fluorescentchelate (EuTDA) and the measured fluorescence is directly proportionalto the number of cells lysed. Percent specific lysis can be calculatedusing the formula: (Experimental lysis-antibody-independentlysis/maximal lysis antibody-independent lysis×100%).

In some embodiments, if the sensitivity of the fluorescence-based ADCCassay is too low to detect ADCC activity of the therapeutic antibodies,the invention encompasses using radioactive-based ADCC assays, such as⁵¹Cr release assay. Radioactive-based assays may be done instead of, orin combination with, fluorescent-based ADCC assays.

An exemplary ⁵¹Cr release assay for characterizing the antibodies of theinvention can comprise the following: labeling 1-2×10⁶ target cells suchas OVCAR-3 cells with ⁵¹Cr; osponizing the target cells with antibodies4D5 and CC49 in the presence and absence of an antibody of the inventionand adding 5×10³ cells to 96 well plate (preferably 4D5 and CC49 are ata concentration varying from 1-15 μg/mL); adding the opsonized targetcells to monocyte-derived macrophages (MDM) (effector cells), preferablyat a ratio varying from 10:1 to 100:1; incubating the mixture of cellsfor 16-18 hours at 37° C.; collecting supernatants; and analyzing theradioactivity in the supernatant. The cytotoxicity of 4D5 and CC49 inthe presence and absence of an antibody of the invention can then bedetermined, for example, using the following formula percent specificlysis=(experimental lysis-antibody independent lysis/maximallysis-antibody independent lysis)×100%.

In some embodiments, the in vivo activity of the FcγRIIB humanizedantibodies of the invention is determined in xenograft human tumormodels. Tumors may be established using any of the cancer cell linesdescribed supra. In some embodiments, the tumors will be establishedwith two cancer cell lines, wherein the first cancer cell line ischaracterized by a low expression of a cancer antigen and a secondcancer cell line, wherein the second cancer cell line is characterizedby a high expression of the same cancer antigen. Tumor clearance maythen be determined using methods known to one skilled in the art, usingan anti-tumor antibody which immunospecifically binds the cancer antigenon the first and second cancer cell line, and an appropriate mousemodel, e.g., a Balb/c nude mouse model (e.g., Jackson Laboratories,Taconic), with adoptively transferred human monocytes and MDMs aseffector cells. Any of the antibodies described supra may then be testedin this animal model to evaluate the role of anti-FcγRIIB antibody ofthe invention in tumor clearance. Mice that may be used in the inventioninclude for example FcγRIII −/− (where FcγRIIIA is knocked out); Fcγ−/−nude mice (where FcγRI and FcγRIIIA are knocked out); or human FcγRIIBknock in mice or a transgenic knock-in mice, where mouse fcgr2 and fcgr3loci on chromosome 1 are inactivated and the mice express human FcγRIIA,human FcγRIIA human FcγRIIB, human FcγRIIC, human FcγRIIIA, and humanFcγRIIIB.

An exemplary method for testing the in vivo activity of an antibody ofthe invention may comprise the following: establishing a xenograftmurine model using a cancer cell line characterized by the expression ofa cancer antigen and determining the effect of an antibody of theinvention on an antibody specific for the cancer antigen expressed inthe cancer cell line in mediating tumor clearance. Preferably, the invivo activity is tested parallel using two cancer cell lines, whereinthe first cancer cell line is characterized by a first cancer antigenexpressed at low levels and a second cancer cell line, characterized bythe same cancer antigen expressed at a higher level relative to thefirst cancer cell line. These experiments will thus increase thestringency of the evaluation of the role of an antibody of the inventionin tumor clearance. For example, tumors may be established with theIGROV-1 cell line and the effect of an anti-FcγRIIB antibody of theinvention in tumor clearance of a Her2/neu specific antibody may beassessed. In order to establish the xenograft tumor models, 5×10⁶ viablecells, e.g., IGROV-1, SKBR3, may be injected, e.g., s.c. into mice,e.g., 8 age and weight matched female nude athymic mice using, forexample, Matrigel (Becton Dickinson). The estimated weight of the tumormay be determined by the formula: length×(width)²/2; and preferably doesnot exceed 3 grams. Injection of IGROV-1 cells s.c. gives rise to fastgrowing tumors while the i.p. route induces a peritoneal carcinomatosiswhich kills mice in 2 months (Benard et al. (1985) “Characterization ofa Human Ovarian Adenocarcinoma Line, IGROV1, in Tissue Culture and inNude Mice,” Cancer Research, 45:4970-4979). Since the IGROV-1 cells formtumors within 5 weeks, at day 1 after tumor cell injection, monocytes aseffectors are co-injected i.p. along with a therapeutic antibodyspecific for Her2/neu, e.g., Ch4D5, and an antibody of the invention;e.g. chimeric 2B6 or 3H7 as described supra. Preferably, the antibodiesare injected at 4 μg each per gram of mouse body weight (mbw). Theinitial injection will be followed by weekly injections of antibodiesfor 4-6 weeks thereafter at 2 μg/wk. Human effector cells will bereplenished once in 2 weeks. A group of mice will receive no therapeuticantibody but will be injected with a chimeric 4D5 comprising a N297Amutation and human IgG1 as isotype control antibodies for the anti-tumorand anti-FcγRIIB antibodies, respectively. Mice may be placed in groupsof 4 and monitored three times weekly.

TABLE 3 below is an exemplary setup for tumor clearance studies inaccordance with the invention. As shown in TABLE 3, six groups of 8 miceeach will be needed for testing the role of an antibody of the inventionin tumor clearance, wherein one target and effector cell combination isused and wherein two different combinations of the antibodyconcentration are used. In group A, only tumor cells are injected; ingroup B, tumor cells and monocytes are injected; in group C, tumorcells, monocytes, an anti-tumor antibody (ch4D5) are injected; in groupD, tumor cells, monocytes, anti-tumor antibody, and an anti-FcγRIIantibody are injected; in group E, tumor cells, monocytes and ananti-FcγRIIB antibody are injected; in group F, tumor cells, monocytes,Ch4D5 (N297Q), and human IgG1 are injected. It will be appreciated byone skilled in the art that various antibody concentrations of variousantibody combinations may be tested in the tumor models described.Preferably, studies using a breast cancer cell line, e.g., SKBR3, iscarried out in parallel to the above-described experiment.

TABLE 3 Exemplary Experimental Set Up In Mice ch4D5 ch2B6 Human ch4D5(N297Q at (N297Q at (IgG1 4 μg/gm 8 mice Tumor (4 μg/gm 4 μg/gm of 4μg/gm of per cell s.c Monocytes of mbw mbw day 1 of mbw mbw day 1 groupday 0 i.p at day 1 day 1 i.p.) i.p.) day 1 i.p.) i.p.) A + − − − − −B + + − − − − C + + + − − − D + + + − + − E + + − − + − F + + − + − +

The endpoint of the xenograft tumor models is determined based on thesize of the tumors, weight of mice, survival time and histochemical andhistopathological examination of the cancer, using methods known to oneskilled in the art. Each of the groups of mice in Table 3 will beevaluated. Mice are preferably monitored three times a week. Criteriafor tumor growth may be abdominal distention, presence of palpable massin the peritoneal cavity. Preferably estimates of tumor weight versusdays after inoculation will be calculated. A comparison of theaforementioned criteria of mice in Group D compared to those in othergroups will define the role of an antibody of the invention inenhancement of tumor clearance. Preferably, antibody-treated animalswill be under observation for an additional 2 months after the controlgroup.

In alternative embodiments, human FcγRIIB “knock in” mice expressinghuman FcγRIIB on murine effector cells may be used in establishing thein vivo activity of the antibodies of the invention, rather thanadoptively transferring effector cells. Founder mice expressing thehuman FcγRIIB may be generated by “knocking in” the human FcγRIIB ontothe mouse FcγRIIB locus. The founders can then be back-crossed onto thenude background and will express the human FcγRIIB receptor. Theresulting murine effector cells will express endogenous activating FcγRIand FcγRIIIA and inhibitory human FcγRIIB receptors.

The in vivo activity of the humanized antibodies of the invention may befurther tested in a xenograft murine model with human primary tumorderived cells, such as human primary ovarian and breast carcinomaderived cells. Ascites and pleural effusion samples from cancer patientsmay be tested for expression of Her2/neu, using methods known to oneskilled in the art. Samples from ovarian carcinoma patients may beprocessed by spinning down the ascites at 6370 g for 20 minutes at 4°C., lysing the red blood cells, and washing the cells with PBS. Once theexpression of Her2/neu in tumor cells is determined, two samples, amedian and a high expresser may be selected for s.c. inoculation toestablish the xenograft tumor model. The isolated tumor cells will thenbe injected i.p. into mice to expand the cells. Approximately 10 micemay be injected i.p. and each mouse ascites further passaged into twomice to obtain ascites from a total of 20 mice which can be used toinject a group of 80 mice. Pleural effusion samples may be processedusing a similar method as ascites. The Her2/neu+ tumor cells frompleural effusion samples may be injected into the upper right and leftmammary pads of the mice.

In some embodiments, if the percentage of neoplastic cells in theascites or pleural effusion samples is low compared to other cellularsubsets, the neoplastic cells may be expanded in vitro. In otherembodiments, tumor cells may be purified using CC49 antibody(anti-TAG-72)-coated magnetic beads as described previously, see, e.g.,Barker et al. (2001) “An Immunomagnetic-Based Method For ThePurification Of Ovarian Cancer Cells From Patient-Derived Ascites,”Gynecol. Oncol. 82:57-63, which is incorporated herein by reference inits entirety. Briefly, magnetic beads coated with CC49 antibody can beused to separate the ovarian tumor cells that will be detached from thebeads by an overnight incubation at 37° C. In some embodiments, if thetumor cells lack the TAG-72 antigen, negative depletion using a cocktailof antibodies, such as those provided by Stem Cell Technologies, Inc.,Canada, may be used to enrich the tumor cells.

In other embodiments, other tumors markers besides Her2/neu may be usedto separate tumor cells obtained from the ascites and pleural effusionsamples from non-tumor cells. In the case of pleural effusion or breasttissue, it has been recently reported that CD44 (an adhesion molecule),B38.1 (a breast/ovarian cancer-specific marker), CD24 (an adhesionmolecule) may be used as markers, see, e.g., Al Hajj, et al. (2003)“Prospective Identification Of Tumorigenic Breast Cancer Cells,” Proc.Natl. Acad. Sci. USA 100:3983-3988; which is incorporated herein byreference in its entirety. Once tumor cells are purified they may beinjected s.c. into mice for expansion.

Preferably, immunohistochemistry and histochemistry is performed onascites and pleural effusion of patients to analyze structuralcharacteristics of the neoplasia. Such methods are known to one skilledin the art and encompassed within the invention. The markers that may bemonitored include for example cytokeratin (to identify ovarianneoplastic and mesothelial cells from inflammatory and mesenchymalcells); calretinin (to separate mesothelial from Her2neu positiveneoplastic cells); and CD45 (to separate inflammatory cells from therest of the cell population in the samples). Additional markers that maybe followed include CD3 (T cells), CD20 (B cells), CD56 (NK cells), andCD14 (monocytes). It will be appreciated by one skilled in the art thatthe immunohistochemistry and histochemistry methods described supra, areanalogously applied to any tumor cell for use in the methods of theinvention. After s.c. inoculation of tumor cells, mice are followed forclinical and anatomical changes. As needed, mice may be necropsied tocorrelate total tumor burden with specific organ localization.

In a specific embodiment, tumors are established using carcinoma celllines such as IGROV-1, OVCAR-8, SK-B, and OVCAR-3 cells and humanovarian carcinoma ascites and pleural effusion from breast cancerpatients. The ascites preferably contain both the effectors and thetumor targets for the antibodies being tested. Human monocytes will betransferred as effectors.

V. Prophylactic and Therapeutic Methods

The present invention encompasses antibody-based therapies which involveadministering one or more of the humanized antibodies of the inventionto an animal, preferably a mammal, and most preferably a human, forpreventing, treating, or ameliorating symptoms associated with adisease, disorder, or infection, associated with aberrant levels oractivity of FcγRIIB and/or treatable by altering immune functionassociated with FcγRIIB activity or enhancing cytotoxic activity of asecond therapeutic antibody or enhancing efficacy of a vaccinecomposition. In some embodiments, therapy by administration of one ormore antibodies of the invention is combined with administration of oneor more therapies such as, but not limited to, chemotherapies, radiationtherapies, hormonal therapies, and/or biologicaltherapies/immunotherapies.

FcγRIIB (CD32B) has been found to be expressed in the following tissuetypes: adipose, b-cell, bone, brain, cartilage, colon, endocrine, eye,fetus, gastrointestinal tract, genitourinary, germ cell, head and neck,kidney, lung, lymph node, lymphoreticular, mammary gland, muscle,nervous, ovary, pancreas, pancreatic islet, pituitary gland, placenta,retina, skin, soft tissue, synovium, and uterus (data collected from theCancer Genome Anatomy Project of the National Cancer Institute). Thus,the humanized antibodies of the invention can be used to agonize orantagonize the activity of FcγRIIB in any of these tissues. For example,FcγRIIB is expressed in the placenta and may play a role in transport ofIgG to the fetus and also in scavenging immune complexes (Lyden et al.(2001) “The Fc Receptor for IgG Expressed in the Villus Endothelium ofHuman Placenta Is Fc{gamma}RIIb2,” J. Immunol. 166:3882-3889). Incertain embodiments of the invention, a humanized FcγRIIB antibody canused as an abortifacient.

Prophylactic and therapeutic compounds of the invention include, but arenot limited to, proteinaceous molecules, including, but not limited to,peptides, polypeptides, proteins, including post-translationallymodified proteins, antibodies, etc.; small molecules (less than 1000daltons), inorganic or organic compounds; nucleic acid moleculesincluding, but not limited to, double-stranded or single-stranded DNA,double-stranded or single-stranded RNA, as well as triple helix nucleicacid molecules. Prophylactic and therapeutic compounds can be derivedfrom any known organism (including, but not limited to, animals, plants,bacteria, fungi, and protista, or viruses) or from a library ofsynthetic molecules.

Humanized antibodies may be provided in pharmaceutically acceptablecompositions as known in the art or as described herein. As detailedbelow, the humanized antibodies of the invention can be used in methodsof treating cancer (particularly to enhance passive immunotherapy orefficacy of a cancer vaccine), autoimmune disease, inflammatorydisorders or allergies (e.g., to enhance efficacy of a vaccine fortreatment of allergy).

Humanized antibodies of the present invention that function as aprophylactic and/or therapeutic agent of a disease, disorder, orinfection can be administered to an animal, preferably, a mammal andmost preferably, a human, to treat, prevent or ameliorate one or moresymptoms associated with the disease, disorder, or infection. Antibodiesof the invention can be administered in combination with one or moreother prophylactic and/or therapeutic agents useful in the treatment,prevention or management of a disease, disorder, or infection associatedwith aberrant levels or activity of FcγRIIB and/or treatable by alteringimmune function associated with FcγRIIB activity. In certainembodiments, one or more antibodies of the invention are administered toa mammal, preferably, a human, concurrently with one or more othertherapeutic agents useful for the treatment of cancer. The term“concurrently” is not limited to the administration of prophylactic ortherapeutic agents at exactly the same time, but rather it is meant thatantibodies of the invention and the other agent are administered to asubject in a sequence and within a time interval such that theantibodies of the invention can act together with the other agent toprovide an increased benefit than if they were administered otherwise.For example, each prophylactic or therapeutic agent may be administeredat the same time or sequentially in any order at different points intime; however, if not administered at the same time, they should beadministered sufficiently close in time so as to provide the desiredtherapeutic or prophylactic effect. Each therapeutic agent can beadministered separately, in any appropriate form and by any suitableroute.

In various embodiments, the prophylactic or therapeutic agents areadministered less than 1 hour apart, at about 1 hour apart, at about 1hour to about 2 hours apart, at about 2 hours to about 3 hours apart, atabout 3 hours to about 4 hours apart, at about 4 hours to about 5 hoursapart, at about 5 hours to about 6 hours apart, at about 6 hours toabout 7 hours apart, at about 7 hours to about 8 hours apart, at about 8hours to about 9 hours apart, at about 9 hours to about 10 hours apart,at about 10 hours to about 11 hours apart, at about 11 hours to about 12hours apart, no more than 24 hours apart or no more than 48 hours apart.In preferred embodiments, two or more components are administered withinthe same patient visit.

The dosage amounts and frequencies of administration provided herein areencompassed by the terms therapeutically effective and prophylacticallyeffective. The dosage and frequency further will typically varyaccording to factors specific for each patient depending on the specifictherapeutic or prophylactic agents administered, the severity and typeof cancer, the route of administration, as well as age, body weight,response, and the past medical history of the patient. Suitable regimenscan be selected by one skilled in the art by considering such factorsand by following, for example, dosages reported in the literature andrecommended in the PHYSICIAN'S DESK REFERENCE (56^(th) ed., 2002).

The humanized antibodies of this invention may also be advantageouslyutilized in combination with other monoclonal or chimeric antibodies, Fcfusion proteins, or with lymphokines, cytokines or hematopoietic growthfactors (such as, e.g., IL-2, IL-3, IL-4, IL-7, IL-10 and TGF-β), whichenhance FcγRIIB, for example, serve to increase the number or activityof effector cells which interact with the antibodies and, increaseimmune response. In certain embodiments, a cytokine is conjugated to ananti-FcγRIIB antibody.

The humanized antibodies of this invention may also be advantageouslyutilized in combination with one or more drugs used to treat a disease,disorder, or infection such as, for example anti-cancer agents,anti-inflammatory agents or anti-viral agents, e.g., as detailed below.

A. Cancers

Humanized antibodies of the invention can be used alone or incombination with other therapeutic antibodies known in the art toprevent, inhibit or reduce the growth of primary tumors or metastasis ofcancerous cells. In one embodiment, humanized antibodies of theinvention can be used in combination with antibodies used in cancerimmunotherapy. The invention encompasses the use of the humanizedantibodies of the invention in combination with another therapeuticantibody to enhance the efficacy of such immunotherapy by increasing thepotency of the therapeutic antibody's effector function, e.g., ADCC,CDC, phagocytosis, opsonization, etc. Although not intending to be boundby a particular mechanism of action antibodies of the invention blockFcγRIIB, preferably on monocytes and macrophages and thus enhance thetherapeutic benefits a clinical efficacy of tumor specific antibodiesby, for example, enhancing clearance of the tumors mediated byactivating FcγRs.

Accordingly, the invention provides methods of preventing or treatingcancer characterized by a cancer antigen, when administered incombination with another antibody that specifically binds a cancerantigen and is cytotoxic. The humanized antibodies of the invention areuseful for prevention or treatment of cancer, particularly inpotentiating the cytotoxic activity of cancer antigen-specifictherapeutic antibodies with cytotoxic activity to enhance tumor cellkilling by the antibodies of the invention and/or enhancing, forexample, ADCC activity or CDC activity of the therapeutic antibodies. Incertain embodiments of the invention, humanized antibodies of theinvention are administered with Fc fusion proteins. In a specificembodiment, a humanized antibody of the invention, when administeredalone or in combination with a cytotoxic therapeutic antibody, inhibitsor reduces the growth of primary tumor or metastasis of cancerous cellsby at least 99%, at least 95%, at least 90%, at least 85%, at least 80%,at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, atleast 40%, at least 45%, at least 35%, at least 30%, at least 25%, atleast 20%, or at least 10% relative to the growth of primary tumor ormetastasis in absence of said antibody of the invention. In a preferredembodiment, humanized antibodies of the invention in combination with acytotoxic therapeutic antibody inhibit or reduce the growth of primarytumor or metastasis of cancer by at least 99%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, at least 50%, at least 45%, at least 40%, at least 45%, at least35%, at least 30%, at least 25%, at least 20%, or at least 10% relativeto the growth or metastasis in absence of said antibodies.

The transition from a normal to a malignant state is a multistep processinvolving genetic and epigenetic changes. In fact, numerous alterationsoccur in the cellular regulatory circuits that facilitate thisprogression which enables tumor cells to evade the commitment toterminal differentiation and quiescence that normally regulate tissuehomeostasis. Certain genes have been implicated in invasiveness andmetastatic potential of cancer cells such as CSF-1 (colony stimulatingfactor 1 or macrophage colony stimulating factor). Although notintending to be bound by a particular mechanism of action, CSF-1 maymediate tumor progression and metastasis by recruiting macrophages tothe tumor site where they promote progression of tumor. It is believedthat macrophages have a trophic role in mediating tumor progression andmetastasis perhaps by the secretion of angiogenic factors, e.g.,thymidine phosphorylase, vascular endothelial-derived growth factor;secretion of growth factors such as epidermal growth factor that couldact as a paracrine factor on tumor cells, and thus promoting tumor cellmigration and invasion into blood vessels. (See, e.g., Lin et al. (2001)“Colony-stimulating Factor 1 Promotes Progression of Mammary Tumors toMalignancy,” J. Exp. Med. 193:727-739; Lin et al. (2002) “The MacrophageGrowth Factor CSF-1 In Mammary Gland Development And Tumor Progression,”Journal of Mammary Gland Biology and Neoplasm 7:147-162; Scholl et al.(1993) “Is Colony-Stimulating Factor-1 A Key Mediator Of Breast CancerInvasion And Metastasis?” Molecular Carcinogenesis, 7:207-211; Clynes etal. (2000) “Inhibitory Fc Receptors Modulate In Vivo Cytoxicity AgainstTumor Targets,” Nature Medicine, 6:443-446; Fidler et al. (1985)“Macrophages And Metastasis—A Biological Approach To Cancer Therapy,”Cancer Research, 45: 4714-4726).

The invention encompasses using the humanized antibodies of theinvention to block macrophage mediated tumor cell progression andmetastasis. The antibodies of the invention are particularly useful inthe treatment of solid tumors, where macrophage infiltration occurs. Theantagonistic antibodies of the invention are particularly useful forcontrolling, e.g., reducing or eliminating, tumor cell metastasis, byreducing or eliminating the population of macrophages that are localizedat the tumor site. In some embodiments, the humanized antibodies of theinvention are used alone to control tumor cell metastasis. Although notintending to be bound by a particular mechanism of action, theantagonistic antibodies of the invention, when administered alone bindthe inhibitory FcγRIIB on macrophages and effectively reduce thepopulation of macrophages and thus restrict tumor cell progression. Theantagonistic antibodies of the invention reduce, or preferably,eliminate macrophages that are localized at the tumor site, sinceFcγRIIB is preferentially expressed on activated monocytes andmacrophages including tumor-infiltrating macrophages. In someembodiments, the humanized antibodies of the invention are used in thetreatment of cancers that are characterized by the overexpression ofCSF-1, including, but not limited to, breast, uterine, and ovariancancers.

The invention further encompasses humanized antibodies that effectivelydeplete or eliminate immune cells other than macrophages that expressFcγRIIB, e.g., dendritic cells and B-cells. Effective depletion orelimination of immune cells using the antibodies of the invention mayrange from a reduction in population of the immune cells by 50%, 60%,70%, 80%, preferably 90%, and most preferably 99%. Thus, the humanizedantibodies of the invention have enhanced therapeutic efficacy eitheralone or in combination with a second antibody, e.g., a therapeuticantibody such as anti-tumor antibodies, anti-viral antibodies, andanti-microbial antibodies. In some embodiments, the therapeuticantibodies have specificity for a cancer cell or an inflammatory cell.In other embodiments, the second antibody binds a normal cell. Althoughnot intending to be bound by a particular mechanism of action, when theantibodies of the invention are used alone to deplete FcγRIIB-expressingimmune cells, the population of cells is redistributed so thateffectively the cells that are remaining have the activating Fcreceptors and thus the suppression by FcγRIIB is alleviated. When usedin combination with a second antibody, e.g., a therapeutic antibody theefficacy of the second antibody is enhanced by increasing theFc-mediated effector function of the antibody.

Cancers and related disorders that can be treated or prevented bymethods and compositions of the present invention include, but are notlimited to, the following: leukemias including, but not limited to,acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemiassuch as myeloblastic, promyelocytic, myelomonocytic, monocytic,erythroleukemia leukemias and myelodysplastic syndrome, chronicleukemias such as but not limited to, chronic myelocytic (granulocytic)leukemia, chronic lymphocytic leukemia, hairy cell leukemia;polycythemia vera; lymphomas such as, but not limited to, Hodgkin'sdisease, non-Hodgkin's disease; multiple myelomas such as, but notlimited to, smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenström's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone and connective tissue sarcomas such as, butnot limited to, bone sarcoma, osteosarcoma, chondrosarcoma, Ewing'ssarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma,periosteal sarcoma, soft-tissue sarcomas, angiosarcoma(hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovialsarcoma; brain tumors including but not limited to, glioma, astrocytoma,brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor,acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma,pineocytoma, pineoblastoma, primary brain lymphoma; breast cancerincluding, but not limited to, adenocarcinoma, lobular (small cell)carcinoma, intraductal carcinoma, medullary breast cancer, mucinousbreast cancer, tubular breast cancer, papillary breast cancer, Paget'sdisease, and inflammatory breast cancer; adrenal cancer, including butnot limited to, pheochromocytom and adrenocortical carcinoma; thyroidcancer such as but not limited to papillary or follicular thyroidcancer, medullary thyroid cancer and anaplastic thyroid cancer;pancreatic cancer, including but not limited to, insulinoma, gastrinoma,glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid orislet cell tumor; pituitary cancers including but not limited to,Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetesinsipius; eye cancers including, but not limited to, ocular melanomasuch as iris melanoma, choroidal melanoma, and cilliary body melanoma,and retinoblastoma; vaginal cancers, including, but not limited to,squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer,including but not limited to, squamous cell carcinoma, melanoma,adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease;cervical cancers including, but not limited to, squamous cell carcinoma,and adenocarcinoma; uterine cancers including, but not limited to,endometrial carcinoma and uterine sarcoma; ovarian cancers including,but not limited to, ovarian epithelial carcinoma, borderline tumor, germcell tumor, and stromal tumor; esophageal cancers including, but notlimited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma,plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma;stomach cancers including, but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; rectal cancers; liver cancers including, but not limited to,hepatocellular carcinoma and hepatoblastoma, gallbladder cancersincluding, but not limited to, adenocarcinoma; cholangiocarcinomasincluding, but not limited to, papillary, nodular, and diffuse; lungcancers including but not limited to, non-small cell lung cancer,squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,large-cell carcinoma and small-cell lung cancer; testicular cancersincluding, but not limited to, germinal tumor, seminoma, anaplastic,classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancersincluding, but not limited to, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers including, but not limitedto, squamous cell carcinoma; basal cancers; salivary gland cancersincluding, but not limited to, adenocarcinoma, mucoepidermoid carcinoma,and adenoidcystic carcinoma; pharynx cancers including, but not limitedto, squamous cell cancer, and verrucous; skin cancers including, but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acral lentiginous melanoma; kidney cancers including, but notlimited to, renal cell cancer, adenocarcinoma, hypemephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);Wilms' tumor; bladder cancers including, but not limited to,transitional cell carcinoma, squamous cell cancer, adenocarcinoma,carcinosarcoma. In addition, cancers include myxosarcoma, osteogenicsarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas (for areview of such disorders, see Fishman et al. (1985) MEDICINE, 2d Ed.,J.B. Lippincott Co., Philadelphia et al. (1997) INFORMED DECISIONS: THECOMPLETE BOOK OF CANCER DIAGNOSIS, TREATMENT, AND RECOVERY, VikingPenguin, Penguin Books U.S.A., Inc., United States of America).

Accordingly, the methods and compositions of the invention are alsouseful in the treatment or prevention of a variety of cancers or otherabnormal proliferative diseases, including (but not limited to) thefollowing: carcinoma, including that of the bladder, breast, colon,kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;including squamous cell carcinoma; hematopoietic tumors of lymphoidlineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including melanoma, seminoma, tetratocarcinoma, neuroblastomaand glioma; tumors of the central and peripheral nervous system,including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors ofmesenchymal origin, including fibrosafcoma, rhabdomyoscarama, andosteosarcoma; and other tumors, including melanoma, xenodermapegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer andteratocarcinoma. It is also contemplated that cancers caused byaberrations in apoptosis would also be treated by the methods andcompositions of the invention. Such cancers may include, but are not belimited to, follicular lymphomas, carcinomas with p53 mutations, hormonedependent tumors of the breast, prostate and ovary, and precancerouslesions such as familial adenomatous polyposis, and myelodysplasticsyndromes. In specific embodiments, malignancy or dysproliferativechanges (such as metaplasias and dysplasias), or hyperproliferativedisorders, are treated or prevented by the methods and compositions ofthe invention in the ovary, bladder, breast, colon, lung, skin,pancreas, or uterus. In other specific embodiments, sarcoma, melanoma,or leukemia is treated or prevented by the methods and compositions ofthe invention.

Cancers associated with the cancer antigens may be treated or preventedby administration of the antibodies of the invention in combination withan antibody that binds the cancer antigen and is cytotoxic. In oneparticular embodiment, the antibodies of the invention enhance theantibody mediated cytotoxic effect of the antibody directed at theparticular cancer antigen. For example, but not by way of limitation,cancers associated with the following cancer antigens may be treated orprevented by the methods and compositions of the invention: KS 1/4pan-carcinoma antigen (Perez et al. (1989) “Isolation AndCharacterization Of A cDNA Encoding The Ks1/4 Epithelial CarcinomaMarker,” J. Immunol. 142:3662-3667; Möller et al. (1991)“Bispecific-Monoclonal-Antibody-Directed Lysis Of Ovarian CarcinomaCells By Activated Human T Lymphocytes,” Cancer Immunol. Immunother.33(4):210-216), ovarian carcinoma antigen (CA125) (Yu et al. (1991)“Coexpression Of Different Antigenic Markers On Moieties That Bear CA125 Determinants,” Cancer Res. 51(2):468 475), prostatic acid phosphate(Tailor et al. (1990) “Nucleotide Sequence Of Human Prostatic AcidPhosphatase Determined From A Full-Length cDNA Clone,” Nucl. Acids Res.18(16):4928), prostate specific antigen (Henttu et al. (1989) “cDNACoding For The Entire Human Prostate Specific Antigen Shows HighHomologies To The Human Tissue Kallikrein Genes,” Biochem. Biophys. Res.Comm. 10(2):903 910; Israeli et al. (1993) “Molecular Cloning Of AComplementary DNA Encoding A Prostate-Specific Membrane Antigen,” CancerRes. 53:227 230), melanoma-associated antigen p97 (Estin et al. (1989)“Transfected Mouse Melanoma Lines That Express Various Levels Of HumanMelanoma-Associated Antigen p97,” J. Natl. Cancer Instit. 81(6):445454), melanoma antigen gp75 (Vijayasardahl et al. (1990) “The MelanomaAntigen Gp75 Is The Human Homologue Of The Mouse B (Brown) Locus GeneProduct,” J. Exp. Med. 171(4):1375 1380), high molecular weight melanomaantigen (HMW-MAA) (Natali et al. (1987) “Immunohistochemical DetectionOf Antigen In Human Primary And Metastatic Melanomas By The MonoclonalAntibody 140.240 And Its Possible Prognostic Significance,” Cancer 59:5563; Mittelman et al. (1990) “Active Specific Immunotherapy In PatientsWith Melanoma. A Clinical Trial With Mouse Antiidiotypic MonoclonalAntibodies Elicited With SyngeneicAnti-High-Molecular-Weight-Melanoma-Associated Antigen MonoclonalAntibodies,” J. Clin. Invest. 86:2136-2144), prostate specific membraneantigen, carcinoembryonic antigen (CEA) (Foon et al. (1995) “ImmuneResponse To The Carcinoembryonic Antigen In Patients Treated With AnAnti-Idiotype Antibody Vaccine,” J. Clin. Invest. 96(1):334-42),polymorphic epithelial mucin antigen, human milk fat globule antigen,Colorectal tumor-associated antigens such as: CEA, TAG-72 (Yokota et al.(1992) “Rapid Tumor Penetration Of A Single-Chain Fv And Comparison WithOther Immunoglobulin Forms,” Cancer Res. 52:3402-3408), CO17-1A(Ragnhammar et al. (1993) “Effect Of Monoclonal Antibody 17-1A AndGM-CSF In Patients With Advanced Colorectal Carcinoma—Long-Lasting,Complete Remissions Can Be Induced,” Int. J. Cancer 53:751-758); GICA19-9 (Herlyn et al. (1982) “Monoclonal Antibody Detection Of ACirculating Tumor-Associated Antigen. I. Presence Of Antigen In Sera OfPatients With Colorectal, Gastric, And Pancreatic Carcinoma,” J. Clin.Immunol. 2:135-140), CTA-1 and LEA, Burkitt's lymphoma antigen-38.13,CD19 (Ghetie et al. (1994) “Anti-CD19 Inhibits The Growth Of HumanB-Cell Tumor Lines In Vitro And Of Daudi Cells In SCID Mice By InducingCell Cycle Arrest,” Blood 83:1329-1336), human B-lymphoma antigen-CD20(Reff et al. (1994) “Depletion Of B Cells In Vivo By A Chimeric MouseHuman Monoclonal Antibody To CD20,” Blood 83:435-445), CD33 (Sgouros etal. (1993) “Modeling And Dosimetry Of Monoclonal Antibody M195(Anti-CD33) In Acute Myelogenous Leukemia,” J. Nucl. Med. 34:422-430),melanoma specific antigens such as ganglioside GD2 (Saleh et al. (1993)“Generation Of A Human Anti-Idiotypic Antibody That Mimics The GD2Antigen,” J. Immunol., 151, 3390-3398), ganglioside GD3 (Shitara et al.(1993) “A Mouse/Human Chimeric Anti-(Ganglioside GD3) Antibody WithEnhanced Antitumor Activities,” Cancer Immunol. Immunother. 36:373-380),ganglioside GM2 (Livingston et al. (1994) “Improved Survival In StageIII Melanoma Patients With GM2 Antibodies: A Randomized Trial OfAdjuvant Vaccination With GM2 Ganglioside,” J. Clin. Oncol.12:1036-1044), ganglioside GM3 (Hoon et al. (1993) “Molecular Cloning OfA Human Monoclonal Antibody Reactive To Ganglioside GM3 Antigen On HumanCancers,” Cancer Res. 53:5244-5250), tumor-specific transplantation typeof cell-surface antigen (TSTA) such as virally-induced tumor antigensincluding T-antigen DNA tumor viruses and envelope antigens of RNA tumorviruses, oncofetal antigen-alpha-fetoprotein such as CEA of colon,bladder tumor oncofetal antigen (Hellström et al. (1985) “MonoclonalAntibodies To Cell Surface Antigens Shared By Chemically Induced MouseBladder Carcinomas,” Cancer. Res. 45:2210-2188), differentiation antigensuch as human lung carcinoma antigen L6, L20 (Hellström et al. (1986)“Monoclonal Mouse Antibodies Raised Against Human Lung Carcinoma,”Cancer Res. 46:3917-3923), antigens of fibrosarcoma, human leukemia Tcell antigen-Gp37 (Bhattacharya-Chatterjee et al. (1988) “IdiotypeVaccines Against Human T Cell Leukemia. II. Generation AndCharacterization Of A Monoclonal Idiotype Cascade (Ab1, Ab2, and Ab3),”J. Immunol. 141(4):1398-1403), neoglycoprotein, sphingolipids, breastcancer antigen such as EGFR (Epidermal growth factor receptor), HER2antigen (p185^(HER2)), polymorphic epithelial mucin (PEM) (Hilkens etal. (1992) “Cell Membrane-Associated Mucins And TheirAdhesion-Modulating Property,” Trends in Biochem. Sci. 17:359-363),malignant human lymphocyte antigen-APO-1 (Trauth et al. (1989)“Monoclonal Antibody-Mediated Tumor Regression By Induction OfApoptosis,” Science 245:301-304), differentiation antigen (Feizi (1985)“Demonstration By Monoclonal Antibodies That Carbohydrate Structures OfGlycoproteins And Glycolipids Are Onco-Developmental Antigens,” Nature314:53-57) such as I antigen found in fetal erthrocytes and primaryendoderm, I(Ma) found in gastric adencarcinomas, M18 and M39 found inbreast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl,VIM-D5, and D₁56-22 found in colorectal cancer, TRA-1-85 (blood groupH), C14 found in colonic adenocarcinoma, F3 found in lungadenocarcinoma, AH6 found in gastric cancer, Y hapten, Le^(y) found inembryonal carcinoma cells, TL5 (blood group A), EGF receptor found inA431 cells, E₁ series (blood group B) found in pancreatic cancer, FC10.2found in embryonal carcinoma cells, gastric adenocarcinoma, CO-514(blood group Le^(a)) found in adenocarcinoma, NS-10 found inadenocarcinomas, CO-43 (blood group Le^(b)), G49, EGF receptor, (bloodgroup ALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 found incolon cancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄found in melanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2, G_(D2),M1:22:25:8 found in embryonal carcinoma cells and SSEA-3, SSEA-4 foundin 4-8-cell stage embryos. In another embodiment, the antigen is a Tcell receptor derived peptide from a cutaneous T cell lymphoma (seeEdelson (1998) “Cutaneous T-Cell Lymphoma: A Model For SelectiveImmunotherapy,” Cancer J Sci Am. 4:62-71).

The humanized antibodies of the invention can be used in combinationwith any therapeutic cancer antibodies known in the art to enhance theefficacy of treatment. For example, the humanized antibodies of theinvention can be used with any of the antibodies in Table 4 that havedemonstrated therapeutic utility in cancer treatment. The humanizedantibodies of the invention enhance the efficacy of treatment of thetherapeutic cancer antibodies by enhancing at least oneantibody-mediated effector function of said therapeutic cancerantibodies. In one particular embodiment, the humanized antibodiesenhance the efficacy of treatment by enhancing the complement dependentcascade of said therapeutic cancer antibodies. In another embodiment ofthe invention, the humanized antibodies of the invention enhance theefficacy of treatment by enhancing the phagocytosis and opsonization ofthe targeted tumor cells. In another embodiment of the invention, thehumanized antibodies of the invention enhance the efficacy of treatmentby enhancing antibody-dependent cell-mediated cytotoxicity (“ADCC”) indestruction of the targeted tumor cells.

Humanized antibodies of the invention can also be used in combinationwith cytosine-guanine dinucleotides (“CpG”)-based products that havebeen developed (Coley Pharmaceuticals) or are currently being developedas activators of innate and acquired immune responses. For example, theinvention encompasses the use of CpG 7909, CpG 8916, CpG 8954 (ColeyPharmaceuticals) in the methods and compositions of the invention forthe treatment and/or prevention of cancer (See also Warren et al. (2002)“Synergism Between Cytosine-Guanine Oligodeoxynucleotides And MonoclonalAntibody In The Treatment Of Lymphoma,” Semin. Oncol., 29(1 Suppl 2):9397; Warren et al. (2000) “CpG Oligodeoxynucleotides Enhance MonoclonalAntibody Therapy Of A Murine Lymphoma,” Clin. Lymphoma, 1(1):57-61,which are incorporated herein by reference).

Humanized antibodies of the invention can be used in combination with atherapeutic antibody that does not mediate its therapeutic effectthrough cell killing to potentiate the antibody's therapeutic activity.In a specific embodiment, the invention encompasses use of theantibodies of the invention in combination with a therapeutic apoptosisinducing antibody with agonistic activity, e.g., an anti-Fas antibody.Anti-Fas antibodies are known in the art and include, for example, Jo2(Ogasawara et al. (1993) “Lethal Effect Of The Anti-Fas Antibody InMice,” Nature 364: 806-809) and HFE7 (Ichikawa et al. (2000) “A NovelMurine Anti-Human Fas mAb Which Mitigates Lymphadenopathy WithoutHepatotoxicity,” Int. Immunol. 12: 555-562). Although not intending tobe bound by a particular mechanisms of action, FcγRIIB has beenimplicated in promoting anti-Fas mediated apoptosis, see, e.g., Xu etal. (2003) “FcRs Modulate Cytotoxicity Of Anti-Fas Antibodies:Implications For Agonistic Antibody-Based Therapeutics,” J. Immunol.171: 562-568. In fact, the extracellular domain of FcγRIIB may serve asa cross-linking agent for Fas receptors, leading to a functional complexand promoting Fas dependent apoptosis. In some embodiments, theantibodies of the invention block the interaction of anti-Fas antibodiesand FcγRIIB, leading to a reduction in Fas-mediated apoptotic activity.Antibodies of the invention that result in a reduction in Fas-mediatedapoptotic activity are particularly useful in combination with anti-Fasantibodies that have undesirable side effects, e.g., hepatotoxicity. Inother embodiments, the antibodies of the invention enhance theinteraction of anti-Fas antibodies and FcγRIIB, leading to anenhancement of Fas-mediated apoptotic activity. Combination of theantibodies of the invention with therapeutic apoptosis inducingantibodies with agonistic activity have an enhanced therapeuticefficacy.

Therapeutic apoptosis inducing antibodies used in the methods of theinvention may be specific for any death receptor known in the art forthe modulation of apoptotic pathway, e.g., TNFR receptor family.

The invention provides a method of treating diseases with impairedapoptotic mediated signaling, e.g., cancer or autoimmune disease. In aspecific embodiment, the invention encompasses a method of treating adisease with deficient Fas-mediated apoptosis, said method comprisingadministering an antibody of the invention in combination with ananti-Fas antibody.

In some embodiments, the agonistic antibodies of the invention areparticularly useful for the treatment of tumors of non-hematopoieticorigin, including tumors of melanoma cells. Although not intending to bebound by a particular mechanism of action, the efficacy of the agonisticantibodies of the invention is due, in part, to activation of FcγRIIBinhibitory pathway, as tumors of non-hematopoietic origin, includingtumors of melanoma cells express FcγRIIB. Recent experiments have infact shown that expression of FcγRIIB in melanoma cells modulates tumorgrowth by direct interaction with anti-tumor antibodies (e.g., bybinding the Fc region of the anti-tumor antibodies) in anintracytoplasmic-dependent manner (Cassard et al. (2002) “Modulation OfTumor Growth By Inhibitory Fc(gamma) Receptor Expressed By HumanMelanoma Cells,” Journal of Clinical Investigation, 110(10): 1549-1557).

In some embodiments, the invention encompasses use of the antibodies ofthe invention in combination with therapeutic antibodies thatimmunospecifically bind to tumor antigens that are not expressed on thetumor cells themselves, but rather on the surrounding reactive and tumorsupporting non-malignant cells comprising the tumor stroma. The tumorstroma comprises endothelial cells forming new blood vessels and stromalfibroblasts surrounding the tumor vasculature. In a specific embodiment,an antibody of the invention is used in combination with an antibodythat immunospecifically binds a tumor antigen on an endothelial cell. Ina preferred embodiment, an antibody of the invention is used incombination with an antibody that immunospecifically binds a tumorantigen on a fibroblast cell, e.g., fibroblast activation protein (FAP).FAP is a 95 KDa homodimeric type II glycoprotein which is highlyexpressed in stromal fibroblasts of many solid tumors, including, butnot limited to, lung, breast, and colorectal carcinomas. (See, e.g.,Scanlan et al. (1994) “Molecular Cloning Of Fibroblast ActivationProtein Alpha, A Member Of The Serine Protease Family SelectivelyExpressed In Stromal Fibroblasts Of Epithelial Cancers,” Proc. Natl.Acad. USA, 91: 5657-5661; Park et al. (1999) “Fibroblast ActivationProtein, A Dual Specificity Serine Protease Expressed In Reactive HumanTumor Stromal Fibroblasts,” J. Biol. Chem., 274: 36505-36512; Rettig etal. (1988) “Cell-Surface Glycoproteins Of Human Sarcomas: DifferentialExpression In Normal And Malignant Tissues And Cultured Cells,” Proc.Natl. Acad. Sci. USA 85: 3110-3114; Garin-Chesa et al. (1990) “CellSurface Glycoprotein Of Reactive Stromal Fibroblasts As A PotentialAntibody Target In Human Epithelial Cancers,” Proc. Natl. Acad. Sci. USA87: 7235-7239). Antibodies that immunospecifically bind FAP are known inthe art and encompassed within the invention, see, e.g., Wuest et al.(2001) “Construction Of A Bispecific Single Chain Antibody ForRecruitment Of Cytotoxic T Cells To The Tumour Stroma Associated AntigenFibroblast Activation Protein,” Journal of Biotechnology, 159-168;Mersmann et al. (2001) “Human Antibody Derivatives Against TheFibroblast Activation Protein For Tumor Stroma Targeting Of Carcinomas,”Int. J. Cancer, 92: 240-248; U.S. Pat. No. 6,455,677; all of which areincorporated herein in by reference in their entireties.

Recently IgE's have been implicated as mediators of tumor growth and, infact, IgE-targeted immediate hypersensitivity and allergic inflammationreactions have been proposed as possible natural mechanisms involved inanti-tumor responses (for a review, see, e.g., Vena et al. (1992)“Allergy-Related Diseases And Cancer: An Inverse Association,” Am.Journal of Epidemiol. 122: 66-74; Eriksson et al. (1995) “A ProspectiveStudy Of Cancer Incidence In A Cohort Examined For Allergy,” Allergy 50:718-722). In fact, a recent study has shown loading tumor cells withIgEs reduces tumor growth, leading in some instances to tumor rejection.According to the study, IgE loaded tumor cells not only possess atherapeutic potential but also confer long term antitumor immunity,including activation of innate immunity effector mechanism and T-cellmediated adaptive immune response, see Reali et al. (2001) “IgEsTargeted On Tumor Cells: Therapeutic Activity And Potential In TheDesign Of Tumor Vaccines,” Cancer Res. 61: 5517-5522; which isincorporated herein by reference in its entirety. The antagonisticantibodies of the invention may be used in the treatment and/orprevention of cancer in combination with administration of IgEs in orderto enhance the efficacy of IgE-mediated cancer therapy. Although notintending to be bound by a particular mechanism of action the antibodiesof the invention enhance the therapeutic efficacy of IgE treatment oftumors, by blocking the inhibitory pathway. The antagonistic antibodiesof the invention may enhance the therapeutic efficacy of IgE mediatedcancer therapy by:

-   -   (i.) enhancing the delay in tumor growth;    -   (ii.) enhancing the decrease in the rate of tumor progression;    -   (iii.) enhancing tumor rejection; or    -   (iv.) enhancing protective immune relative to treatment of        cancer with IgE alone.

Cancer therapies and their dosages, routes of administration andrecommended usage are known in the art and have been described in theliterature, see, e.g., PHYSICIAN'S DESK REFERENCE (56^(th) ed., 2002,which is incorporated herein by reference).

B. B Cell Malignancies

The agonistic antibodies of the invention are useful for treating orpreventing any B cell malignancies, particularly non-Hodgkin's lymphomaand chronic lymphocytic leukemia. Other B-cell malignancies includesmall lymphocytic lymphoma, Burkitt's lymphoma, mantle cell lymphomasdiffuse small cleaved cell lymphomas, most follicular lymphomas and somediffuse large B cell lymphomas (DLBCL). FcγRIIB, is a target forderegulation by chromosomal translocation in malignant lymphoma,particularly in B-cell non-Hodgkin's lymphoma (See Callanan et al.(2000) “The IgG Fc Receptor, FcgammaRIIB, Is A Target For DeregulationBy Chromosomal Translocation In Malignant Lymphoma,” Proc. Natl. Acad.Sci. U.S.A., 97(1):309-314). Thus, the antibodies of the invention areuseful for treating or preventing any chronic lymphocytic leukemia ofthe B cell lineage. Chronic lymphocytic leukemia of the B cell lineageare reviewed by Freedman (See Freedman (1990) “Immunobiology Of ChronicLymphocytic Leukemia,” Hemtaol. Oncol. Clin. North Am. 4:405-429).Although not intending to be bound by any mechanism of action, theagonistic antibodies of the invention inhibit or prevent B cellmalignancies inhibiting B cell proliferation and/or activation. Theinvention also encompasses the use of the agonistic antibodies of theinvention in combination with other therapies known (e.g., chemotherapyand radiotherapy) in the art for the prevention and/or treatment of Bcell malignancies. The invention also encompasses the use of theagonistic antibodies of the invention in combination with otherantibodies known in the art for the treatment and or prevention ofB-cell malignancies. For example, the agonistic antibodies of theinvention can be used in combination with the anti-C22 or anti-CD19antibodies disclosed by Goldenberg et al. (U.S. Pat. No. 6,306,393),anti-CD20 antibodies, anti-CD33 antibodies, or anti-CD52 antibodies.

Antibodies of the invention can also be used in combination with, forexample, ONCOSCINT® (¹¹¹In-satumomab pantedide) (target: CEA), VERLUMA®(⁹⁹Tc-nofetumomab merpentan) (target: GP40), PROSTASCINT® (capromabpentedide) (target: PSMA), CEA-SCAN® (arcitumomab) (target: CEA),RITUXAN® (rituximab) (target: CD20), HERCEPTIN® (trastuzumab) (target:HER-2), CAMPATH® (alemtuzumab (target: CD52), MYLOTARG® (gemtuzumabozogamicin) (target: CD33), LYMPHOCIDE™ (epratuzumab) (target: CD22),LYMPHOCIDE™ Y-90 (⁹⁰Y-epratuzumab, target: CD22) and ZEVALIN®(ibritumomab tiuxetan) (target: CD20).

C. Autoimmune Disease and Inflammatory Diseases

The agonistic antibodies of the invention may be used to treat orprevent autoimmune diseases or inflammatory diseases. The presentinvention provides methods of preventing, treating, or managing one ormore symptoms associated with an autoimmune or inflammatory disorder ina subject, comprising administering to said subject a therapeuticallyeffective amount of the antibodies or fragments thereof of theinvention. The invention also provides methods for preventing, treating,or managing one or more symptoms associated with an inflammatorydisorder in a subject further comprising, administering to said subjecta therapeutically effective amount of one or more anti-inflammatoryagents. The invention also provides methods for preventing, treating, ormanaging one or more symptoms associated with an autoimmune diseasefurther comprising, administering to said subject a therapeuticallyeffective amount of one or more immunomodulatory agents.

The humanized antibodies of the invention can also be used incombination with any of the antibodies known in the art for thetreatment and/or prevention of autoimmune disease or inflammatorydisease. A non-limiting example of the antibodies or Fc fusion proteinsthat are used for the treatment or prevention of inflammatory disordersis presented in TABLE 4A, and a non-limiting example of the antibodiesor Fc fusion proteins that are used for the treatment or prevention ofautoimmune disorder is presented in TABLE 4B. The humanized antibodiesof the invention can for example, enhance the efficacy of treatment ofthe therapeutic antibodies or Fc fusion proteins presented in TABLES 5AAND 5B. For example, but not by way of limitation, the antibodies of theinvention can enhance the immune response in the subject being treatedwith any of the antibodies or Fc fusion proteins in TABLES 5A OR 5B.

Humanized antibodies of the invention can also be used in combinationwith for example but not by way of limitation, ORTHOCLONE OKT3®(muromonab), REOPRO® (abciximab), ZENAPEX® (declizumab), SIMULEC®(basiliximab), RITUXAN® (rituximab), SYNAGIS® (palivizumab), andREMICADE® (infliximab).

Humanized antibodies of the invention can also be used in combinationwith cytosine-guanine dinucleotides (“CpG”)-based products that havebeen developed (Coley Pharmaceuticals) or are currently being developedas activators of innate and acquired immune responses. For example, theinvention encompasses the use of CpG 7909, CpG 8916, CpG 8954 (ColeyPharmaceuticals) in the methods and compositions of the invention forthe treatment and/or prevention of autoimmune or inflammatory disorders(Weeratna et al. (2001) “CpG ODN Can Re-Direct The Th Bias OfEstablished Th2 Immune Responses In Adult And Young Mice,” FEMS ImmunolMed Microbiol., 32(1):65-71, which is incorporated herein by reference).

Examples of autoimmune disorders that may be treated by administeringthe antibodies of the present invention include, but are not limited to,alopecia areata, ankylosing spondylitis, antiphospholipid syndrome,autoimmune Addison's disease, autoimmune diseases of the adrenal gland,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritisand orchitis, autoimmune thrombocytopenia, Behcet's disease, bullouspemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CRESTsyndrome, cold agglutinin disease, Crohn's disease, discoid lupus,essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto'sthyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopeniapurpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupuserthematosus, Ménière's disease, mixed connective tissue disease,multiple sclerosis, type 1 or immune-mediated diabetes mellitus,myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritisnodosa, polychrondritis, polyglandular syndromes, polymyalgiarheumatica, polymyositis and dermatomyositis, primaryagammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriaticarthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoidarthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-mansyndrome, systemic lupus erythematosus, lupus erythematosus, takayasuarteritis, temporal arteristis/giant cell arteritis, ulcerative colitis,uveitis, vasculitides such as dermatitis herpetiformis vasculitis,vitiligo, and Wegener's granulomatosis. Examples of inflammatorydisorders include, but are not limited to, asthma, encephilitis,inflammatory bowel disease, chronic obstructive pulmonary disease(COPD), allergic disorders, septic shock, pulmonary fibrosis,undifferentiated spondyloarthropathy, undifferentiated arthropathy,arthritis, inflammatory osteolysis, and chronic inflammation resultingfrom chronic viral or bacteria infections. As described herein, someautoimmune disorders are associated with an inflammatory condition.Thus, there is overlap between what is considered an autoimmune disorderand an inflammatory disorder. Therefore, some autoimmune disorders mayalso be characterized as inflammatory disorders. Examples ofinflammatory disorders which can be prevented, treated or managed inaccordance with the methods of the invention include, but are notlimited to, asthma, encephilitis, inflammatory bowel disease, chronicobstructive pulmonary disease (COPD), allergic disorders, septic shock,pulmonary fibrosis, undifferentiated spondyloarthropathy,undifferentiated arthropathy, arthritis, inflammatory osteolysis, andchronic inflammation resulting from chronic viral or bacteriainfections.

In certain embodiments of the invention, the humanized antibodies of theinvention may be used to treat an autoimmune disease that is moreprevalent in one sex. For example, the prevalence of Graves' disease inwomen has been associated with expression of FcγRIIB2 (see Estienne etal. (2002) “Androgen-Dependent Expression Of FcgammaRIIB2 By ThyrocytesFrom Patients With Autoimmune Graves' Disease: A Possible Molecular ClueFor Sex Dependence Of Autoimmune Disease,” FASEB J. 16:1087-1092).Accordingly, humanized antibodies of the invention may be used to treat,prevent, ameliorate, or manage Graves' disease.

Humanized antibodies of the invention can also be used to reduce theinflammation experienced by animals, particularly mammals, withinflammatory disorders. In a specific embodiment, an antibody reducesthe inflammation in an animal by at least 99%, at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, at least 50%, at least 45%, at least 40%, at least 45%, at least35%, at least 30%, at least 25%, at least 20%, or at least 10% relativeto the inflammation in an animal in the not administered said antibody.In another embodiment, a combination of antibodies reduce theinflammation in an animal by at least 99%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, atleast 50%, at least 45%, at least 40%, at least 45%, at least 35%, atleast 30%, at least 25%, at least 20%, or at least 10% relative to theinflammation in an animal in not administered said antibodies.

Humanized antibodies of the invention can also be used to prevent therejection of transplants.

TABLE 4A Antibodies for Inflammatory Diseases and Autoimmune DiseasesThat Can Be Used In Combination With The Antibodies Of The InventionAntibody Name Target Antigen Product Type Isotype Indication 5G1.1Complement (C5) Humanised IgG Rheumatoid Arthritis 5G1.1 Complement (C5)Humanised IgG SLE 5G1.1 Complement (C5) Humanised IgG Nephritis 5G1.1-SCComplement (C5) Humanised ScFv Cardiopulmano Bypass 5G1.1-SC Complement(C5) Humanised ScFv Myocardial Infarction 5G1.1-SC Complement (C5)Humanised ScFv Angioplasty ABX-CBL CBL Human GvHD ABX-CBL CD147 MurineIgG Allograft rejection ABX-IL8 IL-8 Human IgG2 Psoriasis Antegren VLA-4Humanised IgG Multiple Sclerosis Anti-CD11a CD11a Humanised IgG1Psoriasis Anti-CD18 CD18 Humanised Fab'2 Myocardial infarction Anti-LFA1CD18 Murine Fab'2 Allograft rejection Antova CD40L Humanised IgGAllograft rejection Antova CD40L Humanised IgG SLE BTI-322 CD2 Rat IgGGvHD, Psoriasis CDP571 TNF-alpha Humanised IgG4 Crohn's CDP571 TNF-alphaHumanised IgG4 Rheumatoid Arthritis CDP850 E-selectin HumanisedPsoriasis Corsevin M Fact VII Chimeric Anticoagulant D2E7 TNF-alphaHuman Rheumatoid Arthritis Hu23F2G CD11/18 Humanised Multiple SclerosisHu23F2G CD11/18 Humanised IgG Stroke IC14 CD14 Toxic shock ICM3 ICAM-3Humanised Psoriasis IDEC-114 CD80 Primatised Psoriasis IDEC-131 CD40LHumanised SLE IDEC-131 CD40L Humanised Multiple Sclerosis IDEC-151 CD4Primatised IgG1 Rheumatoid Arthritis IDEC-152 CD23 PrimatisedAsthma/Allergy Infliximab TNF-alpha Chimeric IgG1 Rheumatoid ArthritisInfliximab TNF-alpha Chimeric IgG1 Crohn's LDP-01 beta2-integrinHumanised IgG Stroke LDP-01 beta2-integrin Humanised IgG Allograftrejection LDP-02 alpha4beta7 Humanised Ulcerative Colitis MAK-195F TNFalpha Murine Fab'2 Toxic shock MDX-33 CD64 (FcR) Human Autoimmunehaematogical disorders MDX-CD4 CD4 Human IgG Rheumatoid ArthritisMEDI-507 CD2 Humanised Psoriasis MEDI-507 CD2 Humanised GvHD OKT4A CD4Humanised IgG Allograft rejection OrthoClone OKT4A CD4 Humanised IgGAutoimmune disease Orthoclone/ CD3 Murine mIgG2a Allograft rejectionanti-CD3 OKT3 RepPro/Abciximab gpIIbIIIa Chimeric Fab Complications ofcoronary angioplasty rhuMab-E25 IgE Humanised IgG1 Asthma/AllergySB-240563 IL5 Humanised Asthma/Allergy SB-240683 IL-4 HumanisedAsthma/Allergy SCH55700 IL-5 Humanised Asthma/Allergy Simulect CD25Chimeric IgG1 Allograft rejection SMART CD3 Humanised Autoimmune diseasea-CD3 SMART CD3 Humanised Allograft rejection a-CD3 SMART a-CD3 CD3Humanised IgG Psoriasis Zenapax CD25 Humanised IgG1 Allograft rejection

TABLE 4B Antibodies for Autoimmune Disorders Antibody Indication TargetAntigen ABX-RB2 antibody to CBL antigen on T cells, B cells and NK cellsfully human antibody from the Xenomouse IL1-ra rheumatoid arthritisrecombinant anti- inflammatory protein sTNF-RI chronic inflammatorydisease soluble tumor necrosis rheumatoid arthritis factor a - receptortype I blocks TNF action 5c8 (Anti CD-40 Phase II trials were halted inOct. 99 CD-40 ligand antibody) examine “adverse events” IDEC 131systemic lupus erythyematous (SLE) anti CD40 humanized IDEC 151rheumatoid arthritis primatized; anti-CD4 IDEC 152 asthma primatized;anti-CD23 IDEC 114 psoriasis primatized anti-CD80 MEDI-507 rheumatoidarthritis; multiple sclerosis anti-CD2 Crohn's disease psoriasis LDP-02(anti-b7 inflammatory bowel disease a4b7 integrin receptor on mAb)Chron's disease white blood cells ulcerative colitis (leukocytes) SMARTAnti- autoimmune disorders Anti-Gamma Interferon Gamma Interferonantibody Verteportin rheumatoid arthritis Thalomid leprosy - approvedfor market Chron's inhibitor of tumor necrosis (thalidomide) diseasefactor alpha (TNF alpha) rheumatoid arthritis SelCIDs (selective highlyspecific inhibitors of cytokine inhibitory phosphodiesterase type 4drugs) enzyme (PDE-4) increases levels of cAMP (cyclic adenosinemonophosphate) activates protein kinase A (PKA) blocks transcriptionfactor NK-kB prevents transcription of TNF-a gene decreases productionof TNF-α IMiDs general autoimmune disorders structural analogues of(immunomodulatory thalidomideinhibit TNF-a drugs) MDX-33 blood disorderscaused by autoimmune monoclonal antibody reactions against FcRIreceptors Idiopathic Thrombocytopenia Purpurea (ITP) autoimmunehemolytic anemia MDX-CD4 treat rheumatoid arthritis and other monoclonalantibody autoimmunity against CD4 receptor molecule VX-497 autoimmunedisorders inhibitor of inosine multiple sclerosis monophosphaterheumatoid arthritis dehydrogenase inflammatory bowel disease (enzymeneeded to make lupus new RNA and DNA psoriasis used in production ofnucleotides needed for lymphocyte proliferation) VX-740 rheumatoidarthritis inhibitor of ICE interleukin-1 beta (converting enzymecontrols pathways leading to aggressive immune response regulatescytokines) VX-745 specific to inflammation inhibitor of P38MAP kinaseinvolved in chemical signaling of mitogen activated protein immuneresponse kinase onset and progression of inflammation Enbrel(etanercept) targets TNF (tumor necrosis factor) IL-8 fully human MABagainst IL-8 (interleukin 8) (blocks IL-8 blocks inflammatory response)5G1.1 rheumatoid arthritis a C5 complement inhibitor pemphigoid(dangerous skin rash) psoriasis lupus Apogen MP4 recombinant antigenselectively destroys disease associated T-cells induces apoptosisT-cells eliminated by programmed cell death no longer attack body's owncells specific apogens target specific T-cells

D. Allergy

The invention provides methods for treating or preventing anIgE-mediated and or FcγRI mediated allergic disorder in a subject inneed thereof, comprising administering to said subject a therapeuticallyeffective amount of the agonistic antibodies or fragments thereof of theinvention. Although not intending to be bound by a particular mechanismof action, antibodies of the invention are useful in inhibitingFcεRI-induced mast cell activation, which contributes to acute and latephase allergic responses (Metcalfe et al. (1997) “Mast Cells,” Physiol.Rev. 77:1033-1079). Preferably, the agonistic antibodies of theinvention have enhanced therapeutic efficacy and/or reduced side effectsin comparison with the conventional methods used in the art for thetreatment and/or prevention of IgE mediated allergic disorders.Conventional methods for the treatment and/or prevention of IgE mediatedallergic disorders include, but are not limited to, anti-inflammatorydrugs (e.g., oral and inhaled corticosteroids for asthma),antihistamines (e.g., for allergic rhinitis and atopic dermatitis),cysteinyl leukotrienes (e.g., for the treatment of asthma); anti-IgEantibodies; and specific immunotherapy or desensitization.

Examples of IgE-mediated allergic responses include, but are not limitedto, asthma, allergic rhinitis, gastrointestinal allergies, eosinophilia,conjunctivitis, atopic dermatitis, urticaria, anaphylaxis, or golmerularnephritis.

The invention encompasses molecules, e.g., immunoglobulins, engineeredto form complexes with FcγRI and human FcγRIIB, i.e., specifically bindFcγRI and human FcγRIIB. Preferably, such molecules have therapeuticefficacy in IgE and FcγRI-mediated disorders. Although not intending tobe bound by a particular mechanism of action, the therapeutic efficacyof these engineered molecules is, in part, due to their ability toinhibit mast cell and basophil function.

In a specific embodiment, molecules that specifically bind FcγRI andhuman FcγRIIB are chimeric fusion proteins comprising a binding site forFcγRI and a binding site for FcγRIIB. Such molecules may be engineeredin accordance with standard recombinant DNA methodologies known to oneskilled in the art. In a preferred specific embodiment, a chimericfusion protein for use in the methods of the invention comprises anF(ab′) single chain of an anti-FcγRIIB monoclonal antibody of theinvention fused to a region used as a bridge to link the huFcγ to theC-terminal region of the F(ab′) single chain of the anti-FcγRIIBmonoclonal antibody. One exemplary chimeric fusion protein for use inthe methods of the invention comprises the following: V_(L)/C_(H)(FcγRIIB)-hinge-V_(H)/C_(H) (FcγRIIB)-LINKER-C_(H)ε2-C_(H)ε3-C_(H)ε4.The linker for the chimeric molecules may be five, ten, preferablyfifteen amino acids in length. The length of the linker may vary toprovide optimal binding of the molecule to both FcγRIIB and FcγRI. In aspecific embodiment, the linker is a 15 amino acid linker, consisting ofthe sequence: (Gly₄Ser)₃. Although not intending to be bound by aparticular mechanism of action, the flexible peptide linker facilitateschain pairing and minimizes possible refolding and it will also allowthe chimeric molecule to reach the two receptors, i.e., FcγRIIB andFcγRI on the cells and cross-link them. Preferably, the chimericmolecule is cloned into a mammalian expression vector, e.g., pCI-neo,with a compatible promoter, e.g., cytomegalovirus promoter. The fusionprotein prepared in accordance with the methods of the invention willcontain the binding site for FcεRI (CHε2CHε3) and for FcγRIIB(VL/CL,-hinge-VH/CH). The nucleic acid encoding the fusion proteinprepared in accordance with the methods of the invention is preferablytransfected into 293 cells and the secreted protein is purified usingcommon methods known in the art.

Binding of the chimeric molecules to both human FcεRI and FcγRIIB may beassessed using common methods known to one skilled in the art fordetermining binding to an FcγR. Preferably, the chimeric molecules ofthe invention have therapeutic efficacy in treating IgE mediateddisorders, for example, by inhibiting antigen-driven degranulation andinhibition of cell activation. The efficacy of the chimeric molecules ofthe invention in blocking IgE driven FcεRI-mediated mast celldegranulation may be determined in transgenic mice, which have beenengineered to express the human FcεRα and human FcγRIIB, prior to theiruse in humans.

The invention provides the use of bispecific antibodies for thetreatment and/or prevention of IgE-mediated and/or FcγRI-mediatedallergic disorders. A bispecific antibody (BsAb) binds to two differentepitopes usually on distinct antigens. BsAbs have potential clinicalutility and they have been used to target viruses, virally infectedcells and bacterial pathogens as well as to deliver thrombolitic agentsto blood clots (Cao (1998) “Bispecific Antibodies As NovelBioconjugates,” Bioconj. Chem. 9: 635-644; Koelemij et al. (1999)“Bispecific Antibodies In Cancer Therapy, From The Laboratory To TheClinic,” J. Immunother., 22, 514-524; Segal et al. (1999) “BispecificAntibodies In Cancer Therapy,” Curr. Opin. Immunol., 11, 558-562). Thetechnology for the production of BsIgG and other related bispecificmolecules is available (see, e.g., Carter et al. (2001) “BispecificHuman IgG By Design,” J. of Immunol. Methods, 248, 7-15; Segal et al.(2001) “Introduction: Bispecific Antibodies,” J. of Immunol. Methods,248, 71-76, which are incorporated herein by reference in theirentirety). The instant invention provides bispecific antibodiescontaining one F(ab′) of the anti-FcγRIIB antibody and one F(ab′) of anavailable monoclonal anti-huIgE antibody which aggregates two receptors,FcγRIIB and FcεRI, on the surface of the same cell. Any methodologyknown in the art and disclosed herein may be employed to generatebispecific antibodies for use in the methods of the invention. In aspecific embodiment, the BsAbs will be produced by chemicallycross-linking F(ab′) fragments of an anti-FcγRIIB antibody and ananti-huIgE antibody as described previously, see, e.g., Glennie et al.,1995, TUMOR IMMUNOBIOLOGY, Oxford University press, Oxford, p. 225;which is incorporated herein by reference in its entirety). The F(ab′)fragments may be produced by limited proteolysis with pepsin and reducedwith mercaptoethanol amine to provide Fab′ fragments with freehinge-region sulfhydryl (SH) groups. The SH group on one of the Fab′(SH) fragments may be alkylated with excess 0-phenylenedimaleimide(0-PDM) to provide a free maleimide group (mal). The two preparationsFab′(mal) and Fab′(SH) may be combined at an appropriate ratio,preferably 1:1 to generate heterodimeric constructs. The BsAbs can bepurified by size exclusion chromatography and characterized by HPLCusing methods known to one skilled in the art.

In particular, the invention encompasses bispecific antibodiescomprising a first heavy chain-light chain pair that binds FcγRIIB withgreater affinity than said heavy chain-light chain pair binds FcγRIIA,and a second heavy chain-light chain pair that binds IgE receptor, withthe provision that said first heavy chain-light chain pair binds FcγRIIBfirst. The bispecific antibodies of the invention can be engineeredusing standard techniques known in the art to ensure that the binding toFcγRIIB precedes the binding to the IgE receptor. It will be understoodby one skilled in the art that it is possible to engineer the bispecificantibodies, for example, such that said bispecific antibodies bindFcγRIIB with greater affinity than said antibodies bind IgE receptor.Additionally, the bispecific antibodies can be engineered by techniquesknown in the art, such that the hinge size of the antibody can beincreased in length, for example, by adding linkers, to provide thebispecific antibodies with flexibility to bind the IgE receptor andFcγRIIB receptor on the same cell.

The antibodies of the invention can also be used in combination withother therapeutic antibodies or drugs known in the art for the treatmentor prevention of IgE-mediated allergic disorders. For example, theantibodies of the invention can be used in combination with any of thefollowing: azelastine (ASTELIN®), beclomethasone dipropionate inhaler(VANCERIL®), beclomethasone dipropionate nasal inhaler/spray(VANCENASE®), Beconase budesonide nasal inhaler/spray (RHINOCORT®),cetirizine (ZYRTEC®), chlorpheniramine, pseudoephedrine, Deconamine(SUDAFED®), cromolyn (NASALCROM®, INTAL®, OPTICROM®), desloratadine(CLARINEX®), fexofenadine and pseudoephedrine (ALEGRA-D®), fexofenadine(ALLEGRA®), flunisolide nasal spray (NASALIDE®), fluticasone propionatenasal inhaler/spray (FLONASE®), fluticasone propionate oral inhaler(FLOVENT®), hydroxyzine (VISTARIL®, ATARAX®), loratadine,pseudoephedrine (CLARITIN-D®), loratadine (CLARITIN®), prednisolone(PREDNISOLONE®, PEDIAPRED® Oral Liquid, MEDROL® prednisone, DELTASONE®,Liquid Predsalmeterol), salmeterol xinafoate (SEREVENT®), triamcinoloneacetonide inhaler (AZMACORT®), triamcinolone acetonide nasalinhaler/spray (NASACORT®, or NASACORT AQ®). Antibodies of the inventioncan be used in combination with cytosine-guanine dinucleotides(“CpG”)-based products that have been developed (Coley Pharmaceuticals)or are currently being developed as activators of innate and acquiredimmune responses. For example, the invention encompasses the use of CpG7909, CpG 8916, CpG 8954 (Coley Pharmaceuticals) in the methods andcompositions of the invention for the treatment and/or prevention ofIgE-mediated allergic disorders (See also Weeratna et al. (2001) “CpGODN Can Re-Direct The Th Bias Of Established Th2 Immune Responses InAdult And Young Mice,” FEMS Immunol Med Microbiol., 32(1):65-71, whichis incorporated herein by reference).

The invention encompasses the use of the humanized antibodies of theinvention in combination with any therapeutic antibodies known in theart for the treatment of allergy disorders, e.g., XOLAIR™ (Omalizumab;Genentech); rhuMAB-E25 (BioWorld Today, Nov. 10, 1998, p. 1; Genentech);CGP-51901 (humanized anti-IgE antibody), etc.

Additionally, the invention encompasses the use of the humanizedantibodies of the invention in combination with other compositions knownin the art for the treatment of allergy disorders. In particular,methods and compositions disclosed in Carson et al. (U.S. Pat. No.6,426,336; US 2002/0035109 A1; US 2002/0010343) is incorporated hereinby reference in its entirety.

E. Immunomodulatory Agents and Anti-Inflammatory Agents

The method of the present invention provides methods of treatment forautoimmune diseases and inflammatory diseases comprising administrationof the antibodies of the present invention in conjunction with othertreatment agents. Examples of immunomodulatory agents include, but arenot limited to, methothrexate, ENBREL® (etanerecept), REMICADE®(infliximab), leflunomide, cyclophosphamide, cyclosporine A, andmacrolide antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone(MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin(sirolimus), mizoribine, deoxyspergualin, brequinar,malononitriloamindes (e.g., leflunamide), T cell receptor modulators,and cytokine receptor modulators.

Anti-inflammatory agents have exhibited success in treatment ofinflammatory and autoimmune disorders and are now a common and astandard treatment for such disorders. Any anti-inflammatory agentwell-known to one of skill in the art can be used in the methods of theinvention. Non-limiting examples of anti-inflammatory agents includenon-steroidal anti-inflammatory drugs (NSAIDs), steroidalanti-inflammatory drugs, beta-agonists, anticholingeric agents, andmethyl xanthines. Examples of NSAIDs include, but are not limited to,aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™),etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™),ketoralac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™),sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™),naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone(RELAFEN™). Such NSAIDs function by inhibiting a cyclooxygenase enzyme(e.g., COX-1 and/or COX-2). Examples of steroidal anti-inflammatorydrugs include, but are not limited to, glucocorticoids, dexamethasone(DECADRON™), cortisone, hydrocortisone, prednisone (DELTASONE™),prednisolone, triamcinolone, azulfidine, and eicosanoids such asprostaglandins, thromboxanes, and leukotrienes.

F. Anti-Cancer Agents and Therapeutic Antibodies

In a specific embodiment, the methods of the invention encompass theadministration of one or more angiogenesis inhibitors such as but notlimited to: Angiostatin (plasminogen fragment); antiangiogenicantithrombin III; Angiozyme; ABT-627; Bay 12-9566; Benefin; Bevacizumab;BMS-275291; cartilage-derived inhibitor (CDI); CAI; CD59 complementfragment; CEP-7055; Col 3; Combretastatin A-4; Endostatin (collagenXVIII fragment); EGFr blockers/inhibitors (IRESSA® (gefitinib), TARCEVA®(erlotinib), ERBITUX® (cetuximab), and VECTIBIX™ (panitumumab;ABX-EGF)); Fibronectin fragment; Gro-beta; Halofuginone; Heparinases;Heparin hexasaccharide fragment; HMV833; Human chorionic gonadotropin(hCG); IM-862; Interferon alpha/beta/gamma; Interferon inducible protein(IP-10); Interleukin-12; Kringle 5 (plasminogen fragment); Marimastat;Metalloproteinase inhibitors (TIMPs); 2-Methoxyestradiol; MMI 270 (CGS27023A); MoAb IMC-1C11; Neovastat; NM-3; Panzem; PI-88; Placentalribonuclease inhibitor; Plasminogen activator inhibitor; Plateletfactor-4 (PF4); Prinomastat; Prolactin 16kD fragment; Proliferin-relatedprotein (PRP); PTK 787/ZK 222594; Retinoids; Solimastat; Squalamine; SS3304; SU 5416; SU6668; SU11248; Tetrahydrocortisol-S;tetrathiomolybdate; thalidomide; Thrombospondin-1 (TSP-1); TNP-470;Transforming growth factor-beta (TGF-β); Vasculostatin; Vasostatin(calreticulin fragment); ZD6126; ZD 6474; farnesyl transferaseinhibitors (FTI); and bisphosphonates.

Anti-cancer agents that can be used in combination with antibodies ofthe invention in the various embodiments of the invention, includingpharmaceutical compositions and dosage forms and kits of the invention,include, but are not limited to: acivicin; aclarubicin; acodazolehydrochloride; acronine; adozelesin; aldesleukin; altretamine;ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;azotomycin; batimastat; benzodepa; bicalutamide; bisantrenehydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;caracemide; carbetimer; carboplatin; carmustine; carubicinhydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine;dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine;dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel;doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifenecitrate; dromostanolone propionate; duazomycin; edatrexate; eflornithinehydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin hydrochloride; erbulozole; esorubicin hydrochloride;estramustine; estramustine phosphate sodium; etanidazole; etoposide;etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine;fenretinide; floxuridine; fludarabine phosphate; fluorouracil;flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabinehydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;ilmofosine; interleukin II (including recombinant interleukin II, orrIL2), interferon alfa-2a; interferon alfa-2b; interferon alfa-n1;interferon alfa-n3; interferon beta-Ia; interferon gamma-Ib; iproplatin;irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolideacetate; liarozole hydrochloride; lometrexol sodium; lomustine;losoxantrone hydrochloride; masoprocol; maytansine; mechlorethaminehydrochloride; megestrol acetate; melengestrol acetate; melphalan;menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin;mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolicacid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel;pegaspargase; peliomycin; pentamustine; peplomycin sulfate;perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride;plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine;procarbazine hydrochloride; puromycin; puromycin hydrochloride;pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride;semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermaniumhydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin;sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantronehydrochloride; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifenecitrate; trestolone acetate; triciribine phosphate; trimetrexate;trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracilmustard; uredepa; vapreotide; verteporfin; vinblastine sulfate;vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate;vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include,but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone;didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine;dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel;docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;eflornithine; elemene; emitefur; epirubicin; epristeride; estramustineanalogue; estrogen agonists; estrogen antagonists; etanidazole;etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;filgrastim; finasteride; flavopiridol; flezelastine; fluasterone;fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane;fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathioneinhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;insulin-like growth factor-1 receptor inhibitor; interferon agonists;interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticanceragent; mycaperoxide B; mycobacterial cell wall extract; myriaporone;N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin;nemorubicin; neridronic acid; neutral endopeptidase; nilutamide;nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn;O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone;ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; prednisone;propyl bis-acridone; prostaglandin J2; proteasome inhibitors; proteinA-based immune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofuran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic;thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroidstimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocenebichloride; topsentin; toremifene; totipotent stem cell factor;translation inhibitors; tretinoin; triacetyluridine; triciribine;trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinaseinhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenitalsinus-derived growth inhibitory factor; urokinase receptor antagonists;vapreotide; variolin B; vector system, erythrocyte gene therapy;velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatinstimalamer. Preferred additional anti-cancer drugs are 5-fluorouraciland leucovorin.

Examples of therapeutic antibodies that can be used in methods of theinvention include but are not limited to HERCEPTIN® (Trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer; REOPRO®(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection; PANOREX™ (edrecolomab)which is a murine anti-17-IA cell surface antigen IgG2a antibody (GlaxoWellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope)IgG antibody (ImClone System); ERBITUX® (cetuximab) which is a chimericanti-EGFR IgG antibody (ImClone System); VITAXIN™ which is a humanizedanti-αVβ3 integrin antibody (Applied Molecular Evolution/MedImmune);CAMPATH® (alemtuzumab) 1H/LDP-03 which is a humanized anti CD52 IgG1antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN® (rituximab) which is achimeric anti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE® (epratuzumab) which is a humanized anti-CD22 IgG antibody(Immunomedics); ICM3 which is a humanized anti-ICAM3 antibody (ICOSPharm); IDEC-114 which is a primatied anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ which is a radiolabelled murine anti-CD20antibody (IDEC/Schering AG); IDEC-131 which is a humanized anti-CD40Lantibody (IDEC/Eisai); IDEC-151 which is a primatized anti-CD4 antibody(IDEC); IDEC-152 which is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 which is a humanized anti-CD3 IgG(Protein Design Lab); 5G1.1 which is a humanized anti-complement factor5 (C5) antibody (Alexion Pharm); Humira® which is a human anti-TNF-αantibody (Abbott Laboratories); CDP870 which is a humanized anti-TNF-αFab fragment (Celltech); IDEC-151 which is a primatized anti-CD4 IgG1antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 which is a humananti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 which is ahumanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 which is ahumanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4Awhich is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA®(ruplizumab) which is a humanized anti-CD40L IgG antibody (Biogen);ANTEGREN® (natalizumab) which is a humanized anti-VLA-4 IgG antibody(Elan); and CAT-152 which is a human anti-TGF-β₂ antibody (Cambridge AbTech).

Other examples of therapeutic antibodies that can be used in combinationwith the antibodies of the invention are presented in Table 5.

TABLE 5 Monoclonal Antibodies For Cancer Therapy That Can Be Used InCombination With The Antibodies Of The Invention Product Disease TargetABX-EGF Cancer EGF receptor OvaRex ovarian cancer tumor antigen CA125BravaRex metastatic cancers tumor antigen MUC1 Theragyn ovarian cancerPEM antigen (pemtumomabytrrium-90) Therex breast cancer PEM antigenblvatuzumab head & neck CD44 cancer Panorex Colorectal cancer 17-1AReoPro PTCA gp IIIb/IIIa ReoPro Acute MI gp IIIb/IIIa ReoPro Ischemicstroke gp IIIb/IIIa Bexocar NHL CD20 MAb, idiotypic 105AD7 colorectalcancer gp72 vaccine Anti-EpCAM cancer Ep-CAM MAb, lung cancer non-smallcell NA lung cancer Herceptin metastatic breast HER-2 cancer Herceptinearly stage breast HER-2 cancer Rituxan Relapsed/refractory CD20low-grade or follicular NHL Rituxan intermediate & CD20 high-grade NHLMAb-VEGF NSCLC, VEGF metastatic MAb-VEGF Colorectal cancer, VEGFmetastatic AMD Fab age-related CD18 macular degeneration E-26 (2^(nd)gen. IgE) allergic asthma & IgE rhinitis Zevalin (Rituxan + low grade ofCD20 yttrium-90) follicular, relapsed or refractory, CD20-positive, B-cell NHL and Rituximab- refractory NHL Cetuximab + innotecan refractoryEGF receptor colorectal carcinoma Cetuximab + cisplatin & newlydiagnosed EGF receptor radiation or recurrent head & neck cancerCetuximab + gemcitabine newly diagnosed EGF receptor metastaticpancreatic carcinoma Cetuximab + cisplatin + recurrent or EGF receptor5FU or Taxol metastatic head & neck cancer Cetuximab + carboplatin +newly diagnosed EGF receptor paclitaxel non-small cell lung carcinomaCetuximab + cisplatin head & neck EGF receptor cancer (extensiveincurable local- regional disease & distant metasteses) Cetuximab +radiation locally advanced EGF receptor head & neck carcinoma BEC2 +Bacillus Calmette small cell lung mimics ganglioside Guerin carcinomaGD3 BEC2 + Bacillus Calmette melanoma mimics ganglioside Guerin GD3IMC-1C11 colorectal cancer VEGF-receptor with liver metastesesnuC242-DM1 Colorectal, gastric, nuC242 and pancreatic cancer LymphoCideNon-Hodgkins CD22 lymphoma LymphoCide Y-90 Non-Hodgkins CD22 lymphomaCEA-Cide metastatic solid CEA tumors CEA-Cide Y-90 metastatic solid CEAtumors CEA-Scan (Tc-99m- colorectal cancer CEA labeled arcitumomab)(radioimaging) CEA-Scan (Tc-99m- Breast cancer CEA labeled arcitumomab)(radioimaging) CEA-Scan (Tc-99m- lung cancer CEA labeled arcitumomab)(radioimaging) CEA-Scan (Tc-99m- intraoperative CEA labeled arcitumomab)tumors (radio imaging) LeukoScan (Tc-99m- soft tissue CEA labeledsulesomab) infection (radioimaging) LymphoScan (Tc-99m- lymphomas CD22labeled) (radioimaging) AFP-Scan (Tc-99m- liver 7 gem-cell AFP labeled)cancers (radioimaging) HumaRAD-HN head & neck NA (+yttrium-90) cancerHumaSPECT colorectal imaging NA MDX-101 (CTLA-4) Prostate and otherCTLA-4 cancers MDX-210 (her-2 Prostate cancer HER-2 overexpression)MDX-210/MAK Cancer HER-2 Vitaxin Cancer αvβ₃ MAb 425 Various cancers EGFreceptor IS-IL-2 Various cancers Ep-CAM Campath (alemtuzumab) chronicCD52 lymphocytic leukemia CD20-streptavidin Non-Hodgkins CD20(+biotin-yttrium 90) lymphoma Avidicin (albumin + metastatic cancer NANRLU13) Oncolym (+iodine-131) Non-Hodgkins HLA-DR 10 beta lymphomaCotara (+iodine-131) unresectable DNA-associated malignant gliomaproteins C215 (+staphylococcal pancreatic cancer NA enterotoxin) MAb,lung/kidney cancer lung & kidney NA cancer nacolomab tafenatox colon &pancreatic NA (C242 + staphylococcal cancer enterotoxin) Nuvion T cellCD3 malignancies SMART M195 AML CD33 SMART 1D10 NHL HLA-DR antigenCEAVac colorectal cancer, CEA advanced TriGem metastatic GD2-gangliosidemelanoma & small cell lung cancer TriAb metastatic breast MUC-1 cancerCEAVac colorectal cancer, CEA advanced TriGem metastatic GD2-gangliosidemelanoma & small cell lung cancer TriAb metastatic breast MUC-1 cancerNovoMAb-G2 Non-Hodgkins NA radiolabeled lymphoma Monopharm C colorectal& SK-1 antigen pancreatic carcinoma GlioMAb-H (+gelonin gliorna,melanoma NA toxin) & neuroblastoma Rituxan Relapsed/refractory CD20low-grade or follicular NHL Rituxan intermediate & CD20 high-grade NHLING-1 adenomcarcinoma Ep-CAM

G. Vaccine Therapy

The invention provides a method for enhancing an immune response to avaccine composition in a subject, said method comprising administeringto said subject a humanized antibody of the invention or a fragmentthereof that specifically binds FcγRIIB with greater affinity than saidantibody or a fragment thereof binds FcγRIIA, and a vaccine composition,wherein said antibody or a fragment thereof enhances the immune responseto said vaccine composition. In one particular embodiment, said antibodyor a fragment thereof enhances the immune response to said vaccinecomposition by enhancing antigen presentation/and or antigen processingof the antigen to which the vaccine is directed at. Any vaccinecomposition known in the art is useful in combination with theantibodies or fragments thereof of the invention.

Although not intending to be bound by a particular mechanism of action,the antibodies of the invention may block activation of FcγRIIB that isexpressed on certain populations and/or types of dendritic cells andthus enhance the activity of such dendritic cells during activevaccination. This enhanced dendritic cell activity may thus result in anenhanced or better response to prophylatic or therapeutic vaccination.

In one embodiment, the invention encompasses the use of the humanizedantibodies of the invention in combination with any cancer vaccine knownin the art, e.g., CANVAXIN™ (Cancer Vax, Corporation, melanoma and coloncancer); ONCOPHAGE® (HSPPC-96; Antigenics; metastatic melanoma);HER-2/neu cancer vaccine, etc. The cancer vaccines used in the methodsand compositions of the invention can be, for example, antigen-specificvaccines, anti-idiotypic vaccines, dendritic cell vaccines, or DNAvaccines. The invention encompasses the use of the antibodies of theinvention with cell-based vaccines as described by Segal et al. (U.S.Pat. No. 6,403,080), which is incorporated herein by reference in itsentirety. The cell based vaccines used in combination with theantibodies of the invention can be either autologous or allogeneic.Briefly, the cancer-based vaccines as described by Segal et al. arebased on Opsonokine™ product by Genitrix, LLC. Opsonokines™ aregenetically engineered cytokines that, when mixed with tumor cells,automatically attach to the surface of the cells. When the “decorated”cells are administered as a vaccine, the cytokine on the cells activatescritical antigen presenting cells in the recipient, while also allowingthe antigen presenting cells to ingest the tumor cells. The antigenpresenting cells are then able to instruct “killer” T cells to find anddestroy similar tumor cells throughout the body. Thus, the Opsonokine™product converts the tumor cells into a potent anti-tumorimmunotherapeutic.

In one embodiment, the invention encompasses the use of the humanizedantibodies of the invention in combination with any allergy vaccineknown in the art. The humanized antibodies of the invention, can beused, for example, in combination with recombinant hybrid moleculescoding for the major timothy grass pollen allergens used for vaccinationagainst grass pollen allergy, as described by Linhart et al. (2002)“Combination Vaccines For The Treatment Of Grass Pollen AllergyConsisting Of Genetically Engineered Hybrid Molecules With IncreasedImmunogenicity,” FASEB Journal, 16(10):1301-1303, which is incorporatedherein by reference in its entirety. In addition the humanizedantibodies of the invention can be used in combination with DNA-basedvaccinations described by Horner et al. (2002) “ImmunostimulatoryDna-Based Therapeutics For Experimental And Clinical Allergy,” Allergy,57 Suppl, 72:24-29, which is incorporated by reference. Antibodies ofthe invention can be used in combination with Bacille Clamett-Guerin(“BCG”) vaccination as described by Choi et al. (2002) “TherapeuticEffects Of Bcg Vaccination In Adult Asthmatic Patients: A Randomized,Controlled Trial,” Ann. Allergy Asthma Immunology, 88(6): 584-591); andBarlan et al. (2002) “The Impact Of In Vivo Calmette-Guérin BacillusAdministration On In Vitro Ige Secretion In Atopic Children,” JournalAsthma, 39(3):239-246, both of which are incorporated herein byreference in entirety, to downregulate IgE secretion. The humanizedantibodies of the invention are useful in treating food allergies. Inparticular, the humanized antibodies of the invention can be used incombination with vaccines or other immunotherapies known in the art (seeHourihane et al. (2002) “Recent Advances In Peanut Allergy,” Curr. Opin.Allergy Clin. Immunol. 2(3):227-231) for the treatment of peanutallergies.

The methods and compositions of the invention can be used in combinationwith vaccines, in which immunity for the antigen(s) is desired. Suchantigens may be any antigen known in the art. The humanized antibodiesof the invention can be used to enhance an immune response, for example,to infectious agents, diseased or abnormal cells such as, but notlimited to, bacteria (e.g., gram positive bacteria, gram negativebacteria, aerobic bacteria, Spirochetes, Mycobacteria, Rickettsias,Chlamydias, etc.), parasites, fungi (e.g., Candida albicans,Aspergillus, etc.), viruses (e.g., DNA viruses, RNA viruses, etc.), ortumors. Viral infections include, but are not limited to, humanimmunodeficiency virus (HIV); hepatitis A virus, hepatitis B virus,hepatitis C virus, hepatitis D virus, or other hepatitis viruses;cytomagaloviruses, herpes simplex virus-1 (-2,-3,-4,-5,-6), humanpapilloma viruses; Respiratory syncytial virus (RSV), Parainfluenzavirus (PIV), Epstein Barr virus, human metapneumovirus (HMPV), influenzavirus, Severe Acute Respiratory Syndrome (SARS) or any other viralinfections.

The invention encompasses methods and vaccine compositions comprisingcombinations of a humanized antibody of the invention, an antigen and acytokine. Preferably, the cytokine is IL-4, IL-10, or TGF-β.

The invention also encompasses the use of the humanized antibodies ofthe invention to enhance a humoral and/or cell mediated response againstthe antigen(s) of the vaccine composition. The invention furtherencompasses the use of the humanized antibodies of the invention toeither prevent or treat a particular disorder, where an enhanced immuneresponse against a particular antigen or antigens is effective to treator prevent the disease or disorder. Such diseases and disorders include,but are not limited to, viral infections, such as HIV, CMV, hepatitis,herpes virus, measles, etc., bacterial infections, fungal and parasiticinfections, cancers, and any other disease or disorder amenable totreatment or prevention by enhancing an immune response against aparticular antigen or antigens.

VI. Compositions and Methods of Administering

The invention provides methods and pharmaceutical compositionscomprising the humanized antibodies of the invention. The invention alsoprovides methods of treatment, prophylaxis, and amelioration of one ormore symptoms associated with a disease, disorder or infection byadministering to a subject an effective amount of a fusion protein or aconjugated molecule of the invention, or a pharmaceutical compositioncomprising a fusion protein or conjugated molecules of the invention. Ina preferred aspect, an antibody or fusion protein or conjugatedmolecule, is substantially purified (i.e., substantially free fromsubstances that limit its effect or produce undesired side effects). Ina specific embodiment, the subject is an animal, preferably a mammalsuch as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.)and a primate (e.g., monkey such as, a cynomolgous monkey and a human).In a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer acomposition comprising humanized antibodies of the invention, e.g.,encapsulation in liposomes, microparticles, microcapsules, recombinantcells capable of expressing the antibody or fusion protein,receptor-mediated endocytosis (See, e.g., Wu et al. (1987)“Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA CarrierSystem,” J. Biol. Chem. 262:4429-4432), construction of a nucleic acidas part of a retroviral or other vector, etc.

In some embodiments, the humanized antibodies of the invention areformulated in liposomes for targeted delivery of the antibodies of theinvention. Liposomes are vesicles comprised of concentrically orderedphospholipid bilayers which encapsulate an aqueous phase. Liposomestypically comprise various types of lipids, phospholipids, and/orsurfactants. The components of liposomes are arranged in a bilayerconfiguration, similar to the lipid arrangement of biological membranes.Liposomes are particularly preferred delivery vehicles due, in part, totheir biocompatibility, low immunogenicity, and low toxicity. Methodsfor preparation of liposomes are known in the art and are encompassedwithin the invention, see, e.g., Eppstein et al. (1985) “BiologicalActivity Of Liposome-Encapsulated Murine Interferon Gamma Is Mediated ByA Cell Membrane Receptor,” Proc. Natl. Acad. Sci. USA, 82: 3688-3692;Hwang et al. (1980) “Hepatic Uptake And Degradation Of UnilamellarSphingomyelin/Cholesterol Liposomes: A Kinetic Study,” Proc. Natl. Acad.Sci. USA, 77: 4030-4034; U.S. Pat. Nos. 4,485,045 and 4,544,545; all ofwhich are incorporated herein by reference in their entirety.

The invention also encompasses methods of preparing liposomes with aprolonged serum half-life, i.e., enhanced circulation time, such asthose disclosed in U.S. Pat. No. 5,013,556. Preferred liposomes used inthe methods of the invention are not rapidly cleared from circulation,i.e., are not taken up into the mononuclear phagocyte system (MPS). Theinvention encompasses sterically stabilized liposomes which are preparedusing common methods known to one skilled in the art. Although notintending to be bound by a particular mechanism of action, stericallystabilized liposomes contain lipid components with bulky and highlyflexible hydrophilic moieties, which reduces the unwanted reaction ofliposomes with serum proteins, reduces opsonization with serumcomponents and reduces recognition by MPS. Sterically stabilizedliposomes are preferably prepared using polyethylene glycol. Forpreparation of liposomes and sterically stabilized liposome see, e.g.,Bendas et al. (2001) “Immunoliposomes: A Promising Approach To TargetingCancer Therapy,” BioDrugs, 15(4): 215-224; Allen et al. (1987) “LargeUnilamellar Liposomes With Low Uptake Into The ReticuloendothelialSystem,” FEBS Lett. 223: 42-46; Klibanov et al. (1990) “AmphipathicPolyethyleneglycols Effectively Prolong The Circulation Time OfLiposomes,” FEBS Lett., 268: 235-237; Blume et al. (1990) “Liposomes ForThe Sustained Drug Release In Vivo,” Biochim. Biophys. Acta., 1029:91-97; Torchilin .et al. (1996) “How Do Polymers Prolong CirculationTime Of Liposomes?,” J. Liposome Res. 6: 99-116; Litzinger et al. (1994)“Effect Of Liposome Size On The Circulation Time And IntraorganDistribution Of Amphipathic Poly(Ethylene Glycol)-Containing Liposomes,”Biochim. Biophys. Acta, 1190: 99-107; Maruyama et al. (1991) “Effect OfMolecular Weight In Amphipathic Polyethyleneglycol On Prolonging TheCirculation Time Of Large Unilamellar Liposomes,” Chem. Pharm. Bull.,39: 1620-1622; Klibanov et al. (1991) “Activity Of AmphipathicPoly(Ethylene Glycol) 5000 To Prolong The Circulation Time Of LiposomesDepends On The Liposome Size And Is Unfavorable For ImmunoliposomeBinding To Target,” Biochim Biophys Acta, 1062; 142-148; Allen et al.(1994) “The Use Of Glycolipids And Hydrophilic Polymers In AvoidingRapid Uptake Of Liposomes By The Mononuclear Phagocyte System,” Adv.Drug Deliv. Rev., 13: 285-309; all of which are incorporated herein byreference in their entirety. The invention also encompasses liposomesthat are adapted for specific organ targeting, see, e.g., U.S. Pat. No.4,544,545, or specific cell targeting, see, e.g., U.S. PatentApplication Publication No. 2005/0074403. Particularly useful liposomesfor use in the compositions and methods of the invention can begenerated by reverse phase evaporation method with a lipid compositioncomprising phosphatidylcholine, cholesterol, and PEG derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. In some embodiments, a fragment of an antibody of theinvention, e.g., F(ab′), may be conjugated to the liposomes usingpreviously described methods, see, e.g., Martin et al. (1982)“Irreversible Coupling Of Immunoglobulin Fragments To PreformedVesicles. An Improved Method For Liposome Targeting,” J. Biol. Chem.257: 286-288, which is incorporated herein by reference in its entirety.

The humanized antibodies of the invention may also be formulated asimmunoliposomes. Immunoliposomes refer to a liposomal composition,wherein an antibody of the invention or a fragment thereof is linked,covalently or non-covalently to the liposomal surface. The chemistry oflinking an antibody to the liposomal surface is known in the art andencompassed within the invention, see, e.g., U.S. Pat. No. 6,787,153;Allen et al., 1995, STEALTH LIPOSOMES, Boca Rotan: CRC Press, 233-44;Hansen et al. (1995) “Attachment Of Antibodies To Sterically StabilizedLiposomes: Evaluation, Comparison And Optimization Of CouplingProcedures,” Biochim. Biophys. Acta, 1239: 133-144; which areincorporated herein by reference in their entirety. In most preferredembodiments, immunoliposomes for use in the methods and compositions ofthe invention are further sterically stabilized. Preferably, thehumanized antibodies of the invention are linked covalently ornon-covalently to a hydrophobic anchor, which is stably rooted in thelipid bilayer of the liposome. Examples of hydrophobic anchors includebut are not limited to phospholipids, e.g., phosoatidylethanolamine(PE), phospahtidylinositol (PI). To achieve a covalent linkage betweenan antibody and a hydrophobic anchor, any of the known biochemicalstrategies in the art may be used, see, e.g., J. Thomas August, ed.,1997, GENE THERAPY: ADVANCES IN PHARMACOLOGY, Volume 40, Academic Press,San Diego, Calif., p. 399-435, which is incorporated herein by referencein its entirety For example, a functional group on an antibody moleculemay react with an active group on a liposome associated hydrophobicanchor, e.g., an amino group of a lysine side chain on an antibody maybe coupled to liposome associated N-glutaryl-phosphatidylethanolamineactivated with water-soluble carbodiimide; or a thiol group of a reducedantibody can be coupled to liposomes via thiol reactive anchors such aspyridylthiopropionyl-phosphatidylethanolamine. See, e.g., Dietrich etal. (1996) “Functional Immobilization Of A DNA-Binding Protein At AMembrane Interface Via Histidine Tag And Synthetic Chelator Lipids,”Biochemistry, 35: 1100-1105; Loughrey et al. (1987) “A Non-CovalentMethod Of Attaching Antibodies To Liposomes,” Biochim. Biophys. Acta,901: 157-160; Martin et al. (1982) “Irreversible Coupling OfImmunoglobulin Fragments To Preformed Vesicles. An Improved Method ForLiposome Targeting,” J. Biol. Chem. 257: 286-288; Martin et al. (1981)“Immunospecific Targeting Of Liposomes To Cells: A Novel And EfficientMethod For Covalent Attachment Of Fab′ Fragments Via Disulfide Bonds,”Biochemistry, 20: 4429-4438; all of which are incorporated herein byreference in their entirety. Although not intending to be bound by aparticular mechanism of action, immunoliposomal formulations comprisingan antibody of the invention are particularly effective as therapeuticagents, since they deliver the antibody to the cytoplasm of the targetcell, i.e., the cell comprising the FcγRIIB receptor to which theantibody binds. The immunoliposomes preferably have an increasedhalf-life in blood, specifically target cells, and can be internalizedinto the cytoplasm of the target cells thereby avoiding loss of thetherapeutic agent or degradation by the endolysosomal pathway.

The invention encompasses immunoliposomes comprising a humanizedantibody of the invention or a fragment thereof. In some embodiments,the immunoliposomes further comprise one or more additional therapeuticagents, such as those disclosed herein.

The immunoliposomal compositions of the invention comprise one or morevesicle forming lipids, an antibody of the invention or a fragment orderivative thereof, and optionally a hydrophilic polymer. Avesicle-forming lipid is preferably a lipid with two hydrocarbon chains,such as acyl chains and a polar head group. Examples of vesicle forminglipids include phospholipids, e.g., phosphatidylcholine,phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol,sphingomyelin, and glycolipids, e.g., cerebrosides, gangliosides.Additional lipids useful in the formulations of the invention are knownto one skilled in the art and encompassed within the invention. In someembodiments, the immunoliposomal compositions further comprise ahydrophilic polymer, e.g., polyethylene glycol, and ganglioside GM1,which increases the serum half-life of the liposome. Methods ofconjugating hydrophilic polymers to liposomes are well known in the artand encompassed within the invention. For a review of immunoliposomesand methods of preparing them, see, e.g., U.S. Patent ApplicationPublication No. 2003/0044407; PCT International Publication No. WO97/38731, Vingerhoeads et al. (1994) “Immunoliposomes In Vivo,”Immunomethods, 4: 259-272; Maruyama (2000) “In Vivo Targeting ByLiposomes,” Biol. Pharm. Bull. 23(7): 791-799; Abra et al. (2002) “TheNext Generation Of Liposome Delivery Systems: Recent Experience WithTumor-Targeted, Sterically-Stabilized Immunoliposomes And Active-LoadingGradients,” Journal J. Liposome Research, Res. 12(1&2): 1-3; Park (2002)“Tumor-Directed Targeting Of Liposomes,” Bioscience Reports, 22(2):267-281; Bendas et al. (2001) “Immunoliposomes: A Promising Approach ToTargeting Cancer Therapy,” BioDrugs, 14(4): 215-224, J. Thomas August,ed., 1997, GENE THERAPY: ADVANCES IN PHARMACOLOGY, Volume 40, AcademicPress, San Diego, Calif., p. 399-435, all of which are incorporatedherein by reference in their entireties.

Methods of administering an antibody of the invention include, but arenot limited to, parenteral administration (e.g., intradermal,intramuscular, intraperitoneal, intravenous and subcutaneous), epidural,and mucosal (e.g., intranasal and oral routes). In a specificembodiment, the antibodies of the invention are administeredintramuscularly, intravenously, or subcutaneously. The compositions maybe administered by any convenient route, for example, by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and maybe administered together with other biologically active agents.Administration can be systemic or local. In addition, pulmonaryadministration can also be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., U.S.Pat. Nos. 6,019,968; 5,985, 20; 5,985,309; 5,934,272; 5,874,064;5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903, eachof which is incorporated herein by reference in its entirety.

The invention also provides that the humanized antibodies of theinvention are packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of antibody. In oneembodiment, the antibodies of the invention are supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted, e.g., with wateror saline to the appropriate concentration for administration to asubject. Preferably, the antibodies of the invention are supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,or at least 75 mg. The lyophilized antibodies of the invention should bestored at between 2 and 8° C. in their original container and theantibodies should be administered within 12 hours, preferably within 6hours, within 5 hours, within 3 hours, or within 1 hour after beingreconstituted. In an alternative embodiment, antibodies of the inventionare supplied in liquid form in a hermetically sealed containerindicating the quantity and concentration of the antibody, fusionprotein, or conjugated molecule. Preferably, the liquid form of theantibodies are supplied in a hermetically sealed container at least 1mg/ml, more preferably at least 2.5 mg/ml, at least 5 mg/ml, at least 8mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least50 mg/ml, at least 100 mg/ml, at least 150 mg/ml, at least 200 mg/ml ofthe antibodies.

The amount of the composition of the invention which will be effectivein the treatment, prevention or amelioration of one or more symptomsassociated with a disorder can be determined by standard clinicaltechniques. The precise dose to be employed in the formulation will alsodepend on the route of administration, and the seriousness of thecondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

For antibodies encompassed by the invention, the dosage administered toa patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's bodyweight. Preferably, the dosage administered to a patient is between0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kgor 0.01 to 0.10 mg/kg of the patient's body weight. Generally, humanantibodies have a longer half-life within the human body than antibodiesfrom other species due to the immune response to the foreignpolypeptides. Thus, lower dosages of human antibodies and less frequentadministration is often possible. Further, the dosage and frequency ofadministration of antibodies of the invention or fragments thereof maybe reduced by enhancing uptake and tissue penetration of the antibodiesby modifications such as, for example, lipidation.

In one embodiment, the dosage of the antibodies of the inventionadministered to a patient are 0.01 mg to 1000 mg/day, when used assingle agent therapy. In another embodiment the antibodies of theinvention are used in combination with other therapeutic compositionsand the dosage administered to a patient are lower than when saidantibodies are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved by, for example, and not by way oflimitation, local infusion, by injection, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering an antibody of the invention, care must be taken touse materials to which the antibody or the fusion protein does notabsorb.

In another embodiment, the compositions can be delivered in a vesicle,in particular a liposome (See Langer (1990) “New Methods Of DrugDelivery,” Science 249:1527-1533; Treat et al., in LIPOSOMES IN THETHERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.3 17-327; see generally ibid.).

In yet another embodiment, the compositions can be delivered in acontrolled release or sustained release system. Any technique known toone of skill in the art can be used to produce sustained releaseformulations comprising one or more antibodies of the invention. See,e.g., U.S. Pat. No. 4,526,938; PCT publication WO 91/05548; PCTpublication WO 96/20698; Ning et al. (1996) “IntratumoralRadioimmunotheraphy Of A Human Colon Cancer Xenograft Using ASustained-Release Gel,” Radiotherapy & Oncology 39:179-189, Song et al.(1995) “Antibody Mediated Lung Targeting of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal. (1997) “Biodegradable Polymeric Carriers for a bFGF Antibody forCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation ofRecombinant Humanized Monoclonal Antibody for Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety. In one embodiment, apump may be used in a controlled release system (See Langer, supra;Sefton (1987) “Implantable Pumps,” CRC Crit. Ref. Biomed. Eng.14:201-240; Buchwald et al. (1980) “Long-Term, Continuous IntravenousHeparin Administration By An Implantable Infusion Pump In AmbulatoryPatients With Recurrent Venous Thrombosis,” Surgery 88:507-516; andSaudek et al. (1989) “A Preliminary Trial Of The ProgrammableImplantable Medication System For Insulin Delivery,” N. Engl. J. Med.321:574-579). In another embodiment, polymeric materials can be used toachieve controlled release of antibodies (see e.g., MEDICAL APPLICATIONSOF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton,Fla. (1974); CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN ANDPERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984); Ranger etal. (1983) “Ranger et al. (1983) “Chemical And Physical Structure OfPolymers as Carriers For Controlled Release Of Bioactive Agents: AReview,” J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; See also Levyet al. (1985) “Inhibition Of Calcification Of Bioprosthetic Heart ValvesBy Local Controlled-Release Diphosphonate,” Science 228:190-192; Duringet al. (1989) “Controlled Release Of Dopamine From A Polymeric BrainImplant: In Vivo Characterization,” Ann. Neurol. 25:351-356; Howard etal. (1989) “Intracerebral Drug Delivery In Rats With Lesion-InducedMemory Deficits,” J. Neurosurg. 7(1):105-112); U.S. Pat. No. 5,679,377;U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S. Pat. No.5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO 99/15154; andPCT Publication No. WO 99/20253). Examples of polymers used in sustainedrelease formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid),poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides(PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol),polyacrylamide, poly(ethylene glycol), polylactides (PLA),poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In yet anotherembodiment, a controlled release system can be placed in proximity ofthe therapeutic target (e.g., the lungs), thus requiring only a fractionof the systemic dose (see, e.g., Goodson, in MEDICAL APPLICATIONS OFCONTROLLED RELEASE, supra, vol. 2, pp. 115-138 (1984)). In anotherembodiment, polymeric compositions useful as controlled release implantsare used according to Dunn et al. (See U.S. Pat. No. 5,945,155). Thisparticular method is based upon the therapeutic effect of the in situcontrolled release of the bioactive material from the polymer system.The implantation can generally occur anywhere within the body of thepatient in need of therapeutic treatment. In another embodiment, anon-polymeric sustained delivery system is used, whereby a non-polymericimplant in the body of the subject is used as a drug delivery system.Upon implantation in the body, the organic solvent of the implant willdissipate, disperse, or leach from the composition into surroundingtissue fluid, and the non-polymeric material will gradually coagulate orprecipitate to form a solid, microporous matrix (See U.S. Pat. No.5,888,533).

Controlled release systems are discussed in the review by Langer (1990)“New Methods Of Drug Delivery,” Science 249:1527-1533. Any techniqueknown to one of skill in the art can be used to produce sustainedrelease formulations comprising one or more therapeutic agents of theinvention. See, e.g., U.S. Pat. No. 4,526,938; International PublicationNos. WO 91/05548 and WO 96/20698; Ning et al. (1996) “IntratumoralRadioimmunotheraphy Of A Human Colon Cancer Xenograft Using ASustained-Release Gel,” Radiotherapy & Oncology 39:179-189; Song et al.(1995) “Antibody Mediated Lung Targeting Of Long-Circulating Emulsions,”PDA Journal of Pharmaceutical Science & Technology 50:372-397; Cleek etal. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody ForCardiovascular Application,” Pro. Int'l. Symp. Control. Rel. Bioact.Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation OfRecombinant Humanized Monoclonal Antibody For Local Delivery,” Proc.Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which isincorporated herein by reference in its entirety.

In a specific embodiment where the composition of the invention is anucleic acid encoding an antibody, the nucleic acid can be administeredin vivo to promote expression of its encoded antibody, by constructingit as part of an appropriate nucleic acid expression vector andadministering it so that it becomes intracellular, e.g., by use of aretroviral vector (See U.S. Pat. No. 4,980,286), or by direct injection,or by use of microparticle bombardment (e.g., a gene gun; Biolistic,Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, or by administering it in linkage to ahomeobox-like peptide which is known to enter the nucleus (See e.g.,Joliot et al. (1991) “Antennapedia Homeobox Peptide Regulates NeuralMorphogenesis,” Proc. Natl. Acad. Sci. USA 88:1864-1868), etc.Alternatively, a nucleic acid can be introduced intracellularly andincorporated within host cell DNA for expression by homologousrecombination.

For antibodies, the therapeutically or prophylactically effective dosageadministered to a subject is typically 0.1 mg/kg to 200 mg/kg of thesubject's body weight. Preferably, the dosage administered to a subjectis between 0.1 mg/kg and 20 mg/kg of the subject's body weight and morepreferably the dosage administered to a subject is between 1 mg/kg to 10mg/kg of the subject's body weight. The dosage and frequency ofadministration of antibodies of the invention may be reduced also byenhancing uptake and tissue penetration (e.g., into the lung) of theantibodies or fusion proteins by modifications such as, for example,lipidation.

Treatment of a subject with a therapeutically or prophylacticallyeffective amount of antibodies of the invention can include a singletreatment or, preferably, can include a series of treatments. In apreferred example, a subject is treated with antibodies of the inventionin the range of between about 0.1 to 30 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. In other embodiments, the pharmaceuticalcompositions of the invention are administered once a day, twice a day,or three times a day. In other embodiments, the pharmaceuticalcompositions are administered once a week, twice a week, once every twoweeks, once a month, once every six weeks, once every two months, twicea year or once per year. It will also be appreciated that the effectivedosage of the antibodies used for treatment may increase or decreaseover the course of a particular treatment.

A. Pharmaceutical Compositions

The compositions of the invention include bulk drug compositions usefulin the manufacture of pharmaceutical compositions (e.g., impure ornon-sterile compositions) and pharmaceutical compositions (i.e.,compositions that are suitable for administration to a subject orpatient) which can be used in the preparation of unit dosage forms. Suchcompositions comprise a prophylactically or therapeutically effectiveamount of a prophylactic and/or therapeutic agent disclosed herein or acombination of those agents and a pharmaceutically acceptable carrier.Preferably, compositions of the invention comprise a prophylactically ortherapeutically effective amount of humanized antibodies of theinvention and a pharmaceutically acceptable carrier.

In one particular embodiment, the pharmaceutical composition comprisesof a therapeutically effective amount of a humanized antibody or afragment thereof that binds FcγRIIB with a greater affinity than saidantibody or a fragment thereof binds FcγRIIA, a cytotoxic antibody thatspecifically binds a cancer antigen, and a pharmaceutically acceptablecarrier. In another embodiment, said pharmaceutical composition furthercomprises one or more anti-cancer agents.

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant(complete and incomplete), excipient, or vehicle with which thetherapeutic is administered. Such pharmaceutical carriers can be sterileliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

Generally, the ingredients of compositions of the invention are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compositions of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include, but are not limited tothose formed with anions such as those derived from hydrochloric,phosphoric, acetic, oxalic, tartaric acids, etc., and those formed withcations such as those derived from sodium, potassium, ammonium, calcium,ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The present invention also provides pharmaceutical compositions and kitscomprising a FcγRIIB antagonist for use in the prevention, treatment,management, or amelioration of a B-cell malignancy, or one or moresymptoms thereof. In particular, the present invention providespharmaceutical compositions and kits comprising a FcγRIIB antagonist, ananalog, derivative or a humanized anti-FcγRIIB antibody or anantigen-binding fragment thereof.

B. Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or fusion proteins, are administered to treat, prevent orameliorate one or more symptoms associated with a disease, disorder, orinfection, by way of gene therapy. Gene therapy refers to therapyperformed by the administration to a subject of an expressed orexpressible nucleic acid. In this embodiment of the invention, thenucleic acids produce their encoded antibody or fusion protein thatmediates a therapeutic or prophylactic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel et al.(1993) “Human Gene Therapy,” Clinical Pharmacy 12:488-505; Wu et al.(1991) “Delivery Systems For Gene Therapy,” Biotherapy 3:87-95;Tolstoshev (1993) “Gene Therapy, Concepts, Current Trials And FutureDirections,” Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan (1993)“The Basic Science Of Gene Therapy,” Science 260:926-932; and Morgan etal. (1993) “Human Gene Therapy,” Ann. Rev. Biochem. 62:191-217. Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley & Sons, NY (1993); and Kriegler, GENETRANSFER AND EXPRESSION, A LABORATORY MANUAL, Stockton Press, NY (1990).

In a preferred aspect, a composition of the invention comprises nucleicacids encoding an antibody, said nucleic acids being part of anexpression vector that expresses the antibody in a suitable host. Inparticular, such nucleic acids have promoters, preferably heterologouspromoters, operably linked to the antibody coding region, said promoterbeing inducible or constitutive, and, optionally, tissue-specific. Inanother particular embodiment, nucleic acid molecules are used in whichthe antibody coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe antibody encoding nucleic acids (Koller et al. (1989) “InactivatingThe Beta 2-Microglobulin Locus In Mouse Embryonic Stem Cells ByHomologous Recombination,” Proc. Natl. Acad. Sci. USA 86:8932-8935; andZijlstra et al. (1989) “Germ-Line Transmission Of A Disrupted B2Microglobulin Gene Produced By Homologous Recombination In EmbryonicStem Cells,” Nature 342:435-438).

In another preferred aspect, a composition of the invention comprisesnucleic acids encoding a fusion protein, said nucleic acids being a partof an expression vector that expression the fusion protein in a suitablehost. In particular, such nucleic acids have promoters, preferablyheterologous promoters, operably linked to the coding region of a fusionprotein, said promoter being inducible or constitutive, and optionally,tissue-specific. In another particular embodiment, nucleic acidmolecules are used in which the coding sequence of the fusion proteinand any other desired sequences are flanked by regions that promotehomologous recombination at a desired site in the genome, thus providingfor intrachromosomal expression of the fusion protein encoding nucleicacids.

Delivery of the nucleic acids into a subject may be either direct, inwhich case the subject is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the subject. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretroviral or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or by coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (See, e.g., Wu et al. (1987) “Receptor-Mediated In VitroGene Transformation By A Soluble DNA Carrier System,” J. Biol. Chem.262:4429-4432) (which can be used to target cell types specificallyexpressing the receptors), etc. In another embodiment, nucleicacid-ligand complexes can be formed in which the ligand comprises afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (See, e.g., U.S. Patent ApplicationPublication No. 2005/0002903; PCT Publications WO 92/06180; WO 92/22635;WO 92/20316; WO 93/14188; WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller et al. (1989)“Inactivating The Beta 2-Microglobulin Locus In Mouse Embryonic StemCells By Homologous Recombination,” Proc. Natl. Acad. Sci. USA86:8932-8935; and Zijlstra et al. (1989) “Germ-Line Transmission Of ADisrupted B2 Microglobulin Gene Produced By Homologous Recombination InEmbryonic Stem Cells,” Nature 342:435-438).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody or a fusion protein are used. Forexample, a retroviral vector can be used (See Miller et al. (1993) “UseOf Retroviral Vectors For Gene Transfer And Expression,” Meth. Enzymol.217:581-599). These retroviral vectors contain the components necessaryfor the correct packaging of the viral genome and integration into thehost cell DNA. The nucleic acid sequences encoding the antibody or afusion protein to be used in gene therapy are cloned into one or morevectors, which facilitate delivery of the nucleotide sequence into asubject. More detail about retroviral vectors can be found in Boesen etal. (1994) “Circumvention Of Chemotherapy-Induced Myelosuppression ByTransfer Of The mdr1 Gene,” Biotherapy 6:291-302, which describes theuse of a retroviral vector to deliver the mdr 1 gene to hematopoieticstem cells in order to make the stem cells more resistant tochemotherapy. Other references illustrating the use of retroviralvectors in gene therapy include: Clowes et al. (1994) “Long-TermBiological Response Of Injured Rat Carotid Artery Seeded With SmoothMuscle Cells Expressing Retrovirally Introduced Human Genes,” J. Clin.Invest. 93:644-651; Keim et al. (1994) “Retrovirus-Mediated GeneTransduction Into Canine Peripheral Blood Repopulating Cells,” Blood83:1467-1473; Salmons et al. (1993) “Targeting Of Retroviral Vectors ForGene Therapy,” Human Gene Therapy 4:129-141; and Grossman et al. (1993)“Retroviruses: Delivery Vehicle To The Liver,” Curr. Opin. in Geneticsand Devel. 3:110-114.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. For a review ofadenovirus-based gene therapy see Kozarsky et al. (1993) “Gene Therapy:Adenovirus Vectors,” Current Opinion in Genetics and Development3:499-503. The use of adenovirus vectors to transfer genes to therespiratory epithelia of rhesus monkeys has been demonstrated by Bout etal. (1994) “Lung Gene Therapy: In Vivo Adenovirus-Mediated Gene TransferTo Rhesus Monkey Airway Epithelium,” Human Gene Therapy 5:3-10 Otherinstances of the use of adenoviruses in gene therapy can be found inRosenfeld et al. (1991) “Adenovirus-Mediated Transfer Of A RecombinantAlpha 1-Antitrypsin Gene To The Lung Epithelium In Vivo,” Science252:431-434; Rosenfeld et al. (1992) “In Vivo Transfer Of The HumanCystic Fibrosis Transmembrane Conductance Regulator Gene To The AirwayEpithelium,” Cell 68:143-155; Mastrangeli et al. (1993) “Diversity OfAirway Epithelial Cell Targets For In Vivo RecombinantAdenovirus-Mediated Gene Transfer,” J. Clin. Invest. 91:225-234; PCTPublication W094/12649; and Wang et al. (1995) “A Packaging Cell LineFor Propagation Of Recombinant Adenovirus Vectors Containing Two LethalGene-Region Deletions,” Gene Therapy 2:775-783. In a preferredembodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (see, e.g., Walsh et al. (1993) “Gene Therapy For HumanHemoglobinopathies,” Proc. Soc. Exp. Biol. Med. 204:289-300).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to, transfection, electroporation,microinjection, infection with a viral or bacteriophage vector,containing the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcellmediated gene transfer, spheroplast fusion, etc.Numerous techniques are known in the art for the introduction of foreigngenes into cells (See, e.g., Loeffler et al. (1993) “Gene Transfer IntoPrimary And Established Mammalian Cell Lines With Lipopolyamine-CoatedDNA,” Meth. Enzymol. 217:599-618, Cotten et al. (1993)“Receptor-Mediated Transport Of DNA Into Eukaryotic Cells,” Meth.Enzymol. 217:618-644) and may be used in accordance with the presentinvention, provided that the necessary developmental and physiologicalfunctions of the recipient cells are not disrupted. The technique shouldprovide for the stable transfer of the nucleic acid to the cell, so thatthe nucleic acid is expressible by the cell and preferably heritable andexpressible by its cell progeny.

The resulting recombinant cells can be delivered to a subject by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the subject.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody or a fusion protein areintroduced into the cells such that they are expressible by the cells ortheir progeny, and the recombinant cells are then administered in vivofor therapeutic effect. In a specific embodiment, stem or progenitorcells are used. Any stem and/or progenitor cells which can be isolatedand maintained in vitro can potentially be used in accordance with thisembodiment of the present invention (See e.g., PCT Publication WO94/08598; Stemple et al. (1992) “Isolation Of A Stem Cell For NeuronsAnd Glia From The Mammalian Neural Crest,” Cell 7(1):973-985; Rheinwald(1980) “Serial Cultivation Of Normal Human Epidermal Keratinocytes,”Meth. Cell Bio. 21A:229-254; and Pittelkow et al. (1986) “New TechniquesFor The In Vitro Culture Of Human Skin Keratinocytes And Perspectives OnTheir Use For Grafting Of Patients With Extensive Burns,” Mayo ClinicProc. 61:771-777).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

C. Kits

The invention provides a pharmaceutical pack or kit comprising one ormore containers filled with humanized antibodies of the invention.Additionally, one or more other prophylactic or therapeutic agentsuseful for the treatment of a disease can also be included in thepharmaceutical pack or kit. The invention also provides a pharmaceuticalpack or kit comprising one or more containers filled with one or more ofthe ingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises one or more humanizedantibodies of the invention. In another embodiment, a kit furthercomprises one or more other prophylactic or therapeutic agents usefulfor the treatment of cancer, in one or more containers. In anotherembodiment, a kit further comprises one or more cytotoxic antibodiesthat bind one or more cancer antigens associated with cancer. In certainembodiments, the other prophylactic or therapeutic agent is achemotherapeutic. In other embodiments, the prophylactic or therapeuticagent is a biological or hormonal therapeutic.

VII. Characterization and Demonstration of Therapeutic Utility

Several aspects of the pharmaceutical compositions or prophylactic ortherapeutic agents of the invention are preferably tested in vitro,e.g., in a cell culture system, and then in vivo, e.g., in an animalmodel organism, such as a rodent animal model system, for the desiredtherapeutic activity prior to use in humans. For example, assays whichcan be used to determine whether administration of a specificpharmaceutical composition is indicated, include cell culture assays inwhich a patient tissue sample is grown in culture, and exposed to orotherwise contacted with a pharmaceutical composition, and the effect ofsuch composition upon the tissue sample is observed, e.g., inhibition ofor decrease in growth and/or colony formation in soft agar or tubularnetwork formation in three-dimensional basement membrane orextracellular matrix preparation. The tissue sample can be obtained bybiopsy from the patient. This test allows the identification of thetherapeutically most effective prophylactic or therapeutic molecule(s)for each individual patient. Alternatively, instead of culturing cellsfrom a patient, therapeutic agents and methods may be screened usingcells of a tumor or malignant cell line. In various specificembodiments, in vitro assays can be carried out with representativecells of cell types involved in an autoimmune or inflammatory disorder(e.g., T cells), to determine if a pharmaceutical composition of theinvention has a desired effect upon such cell types. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc. Additional assays include raftassociation, CDC, ADCC and apoptosis assays as known in the art anddescribed in the Examples.

Combinations of prophylactic and/or therapeutic agents can be tested insuitable animal model systems prior to use in humans. Such animal modelsystems include, but are not limited to, rats, mice, chicken, cows,monkeys, pigs, dogs, rabbits, etc. Any animal system well-known in theart may be used. In a specific embodiment of the invention, combinationsof prophylactic and/or therapeutic agents are tested in a mouse modelsystem. Such model systems are widely used and well-known to the skilledartisan. Prophylactic and/or therapeutic agents can be administeredrepeatedly. Several aspects of the procedure may vary such as thetemporal regime of administering the prophylactic and/or therapeuticagents, and whether such agents are administered separately or as anadmixture.

Preferred animal models for use in the methods of the invention are forexample, transgenic mice expressing FcγR on mouse effector cells, e.g.,any mouse model described in U.S. Pat. No. 5,877,396 (which isincorporated herein by reference in its entirety). Transgenic mice foruse in the methods of the invention include but are not limited to micecarrying human FcγRIIIA, mice carrying human FcγRIIA, mice carryinghuman FcγRIIB and human FcγRIIIA, mice carrying human FcγRIIB and humanFcγRIIA.

Once the prophylactic and/or therapeutic agents of the invention havebeen tested in an animal model they can be tested in clinical trials toestablish their efficacy. Establishing clinical trials will be done inaccordance with common methodologies known to one skilled in the art,and the optimal dosages and routes of administration as well as toxicityprofiles of the compositions of the invention can be established usingroutine experimentation.

The anti-inflammatory activity of the combination therapies of inventioncan be determined by using various experimental animal models ofinflammatory arthritis known in the art and described in Crofford andWilder, (1993) “Arthritis and Autoimmunity in Animals”, in ARTHRITIS ANDALLIED CONDITIONS: A TEXTBOOK OF RHEUMATOLOGY, McCarty et al. (eds.),Chapter 30. Experimental and spontaneous animal models of inflammatoryarthritis and autoimmune rheumatic diseases can also be used to assessthe anti-inflammatory activity of the combination therapies ofinvention. The following are some assays provided as examples, and notby limitation.

The principle animal models for arthritis or inflammatory disease knownin the art and widely used include: adjuvant-induced arthritis ratmodels, collagen-induced arthritis rat and mouse models andantigen-induced arthritis rat, rabbit and hamster models, all describedin Crofford and Wilder, (1993) “Arthritis and Autoimmunity in Animals”,in ARTHRITIS AND ALLIED CONDITIONS: A TEXTBOOK OF RHEUMATOLOGY, McCartyet al. (eds.), Chapter 30, incorporated herein by reference in itsentirety.

The anti-inflammatory activity of the combination therapies of inventioncan be assessed using a carrageenan-induced arthritis rat model.Carrageenan-induced arthritis has also been used in rabbit, dog and pigin studies of chronic arthritis or inflammation. Quantitativehistomorphometric assessment is used to determine therapeutic efficacy.The methods for using such a carrageenan-induced arthritis model isdescribed in Hansra et al. (2000) “Carrageenan-Induced Arthritis In TheRat,” Inflammation, 24(2): 141-155. Also commonly used arezymosan-induced inflammation animal models as known and described in theart.

The anti-inflammatory activity of the combination therapies of inventioncan also be assessed by measuring the inhibition of carrageenan-inducedpaw edema in the rat, using a modification of the method described inWinter et al. (1962) “Carrageenan-Induced Edema In Hind Paw Of The RatAs An Assay For Anti-Inflammatory Drugs” Proc. Soc. Exp. Biol Med. 111,544-547. This assay has been used as a primary in vivo screen for theanti-inflammatory activity of most NSAIDs, and is considered predictiveof human efficacy. The anti-inflammatory activity of the testprophylactic or therapeutic agents is expressed as the percentinhibition of the increase in hind paw weight of the test group relativeto the vehicle dosed control group.

Additionally, animal models for inflammatory bowel disease can also beused to assess the efficacy of the combination therapies of invention(Kim et al. (1992) “Experimental Colitis In Animal Models,” Scand. J.Gastroentrol. 27:529-537; Strober (1985) “Animal Models Of InflammatoryBowel Disease—An Overview,” Dig. Dis. Sci. 30(12 Suppl):3S-10S).Ulcerative cholitis and Crohn's disease are human inflammatory boweldiseases that can be induced in animals. Sulfated polysaccharidesincluding, but not limited to amylopectin, carrageen, amylopectinsulfate, and dextran sulfate or chemical irritants including but notlimited to trinitrobenzenesulphonic acid (TNBS) and acetic acid can beadministered to animals orally to induce inflammatory bowel diseases.

Animal models for asthma can also be used to assess the efficacy of thecombination therapies of invention. An example of one such model is themurine adoptive transfer model in which aeroallergen provocation of TH1or TH2 recipient mice results in TH effector cell migration to theairways and is associated with an intense neutrophilic (TH1) andeosinophilic (TH2) lung mucosal inflammatory response (Cohn et al.(1997) “Induction Of Airway Mucus Production By T helper 2 (Th2) Cells:A Critical Role For Interleukin 4 In Cell Recruitment But Not MucusProduction,” J. Exp. Med. 186:1737-1747).

Animal models for autoimmune disorders can also be used to assess theefficacy of the combination therapies of invention. Animal models forautoimmune disorders such as type 1 diabetes, thyroid autoimmunity,systemic lupus eruthematosus, and glomerulonephritis have been developed(Flanders et al. (1999) “Prevention Of Type 1 Diabetes From LaboratoryTo Public Health,” Autoimmunity 29:235-246; Rasmussen et al. (1999)“Models To Study The Pathogenesis Of Thyroid Autoimmunity,” Biochimie81:511-515; Foster (1999) “Relevance Of Systemic Lupus ErythematosusNephritis Animal Models To Human Disease,” Semin. Nephrol. 19:12-24).

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for autoimmune and/orinflammatory diseases.

Toxicity and efficacy of the prophylactic and/or therapeutic protocolsof the instant invention can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., fordetermining the LD₅₀ (the dose lethal to 50% of the population) and theED₅₀ (the dose therapeutically effective in 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex and it can be expressed as the ratio LD₅₀/ED₅₀. Prophylacticand/or therapeutic agents that exhibit large therapeutic indices arepreferred. While prophylactic and/or therapeutic agents that exhibittoxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage of the prophylactic and/ortherapeutic agents for use in humans. The dosage of such agents liespreferably within a range of circulating concentrations that include theED₅₀ with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized. For any agent used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

The anti-cancer activity of the therapies used in accordance with thepresent invention also can be determined by using various experimentalanimal models for the study of cancer such as the SCID mouse model ortransgenic mice or nude mice with human xenografts, animal models, suchas hamsters, rabbits, etc. known in the art and described in RELEVANCEOF TUMOR MODELS FOR ANTICANCER DRUG DEVELOPMENT (1999) Fiebig andBurger, eds.); CONTRIBUTIONS TO ONCOLOGY (1999) Karger; THE NUDE MOUSEIN ONCOLOGY RESEARCH (1991) Boven and Winograd, eds.; and ANTICANCERDRUG DEVELOPMENT GUIDE (1997) Teicher, ed., herein incorporated byreference in their entireties.

The protocols and compositions of the invention are preferably tested invitro, and then in vivo, for the desired therapeutic or prophylacticactivity, prior to use in humans. Therapeutic agents and methods may bescreened using cells of a tumor or malignant cell line. Many assaysstandard in the art can be used to assess such survival and/or growth;for example, cell proliferation can be assayed by measuring ³H-thymidineincorporation, by direct cell count, by detecting changes intranscriptional activity of known genes such as proto-oncogenes (e.g.,fos, myc) or cell cycle markers; cell viability can be assessed bytrypan blue staining, differentiation can be assessed visually based onchanges in morphology, decreased growth and/or colony formation in softagar or tubular network formation in three-dimensional basement membraneor extracellular matrix preparation, etc.

Compounds for use in therapy can be tested in suitable animal modelsystems prior to testing in humans, including but not limited to inrats, mice, chicken, cows, monkeys, rabbits, hamsters, etc., forexample, the animal models described above. The compounds can then beused in the appropriate clinical trials.

Further, any assays known to those skilled in the art can be used toevaluate the prophylactic and/or therapeutic utility of thecombinatorial therapies disclosed herein for treatment or prevention ofcancer, inflammatory disorder, or autoimmune disease.

VIII. Diagnostic Methods

Labeled antibodies of the invention can be used for diagnostic purposesto detect, diagnose, or monitor diseases, disorders or infections. Theinvention provides for the detection or diagnosis of a disease, disorderor infection, particularly an autoimmune disease comprising:

-   -   (A) assaying the expression of FcγRIIB in cells or a tissue        sample of a subject using one or more antibodies that        immunospecifically bind to FcγRIIB; and    -   (B) comparing the level of the antigen with a control level,        e.g., levels in normal tissue samples, whereby an increase in        the assayed level of antigen compared to the control level of        the antigen is indicative of the disease, disorder or infection.

Antibodies of the invention can be used to assay FcγRIIB levels in abiological sample using classical immunohistological methods asdescribed herein or as known to those of skill in the art (e.g., seeJalkanen et al. (1985) “Heparan Sulfate Proteoglycans From Mouse MammaryEpithelial Cells: Localization On The Cell Surface With A MonoclonalAntibody,” J. Cell. Biol. 101:976-984; Jalkanen et al. (1987) “CellSurface Proteoglycan Of Mouse Mammary Epithelial Cells Is Shed ByCleavage Of Its Matrix-Binding Ectodomain From Its Membrane-AssociatedDomain,” J. Cell. Biol. 105:3087-3096). Other antibody-based methodsuseful for detecting protein gene expression include immunoassays, suchas the enzyme linked immunosorbent assay (ELISA) and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, alkaline phosphatase, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹³¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium (^(99m)Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine.

One aspect of the invention is the detection and diagnosis of a disease,disorder, or infection in a human. In one embodiment, diagnosiscomprises:

-   -   (A) administering (for example, parenterally, subcutaneously, or        intraperitoneally) to a subject an effective amount of a labeled        antibody that immunospecifically binds to FcγRIIB;    -   (B) waiting for a time interval following the administration for        permitting the labeled antibody to preferentially concentrate at        sites in the subject where FcγRIIB is expressed (and for unbound        labeled molecule to be cleared to background level);    -   (C) determining background level; and    -   (D) detecting the labeled antibody in the subject, such that        detection of labeled antibody above the background level        indicates that the subject has the disease, disorder, or        infection.        In accordance with this embodiment, the antibody is labeled with        an imaging moiety which is detectable using an imaging system        known to one of skill in the art. Background level can be        determined by various methods including, comparing the amount of        labeled molecule detected to a standard value previously        determined for a particular system.

It is understood in the art that the size of the subject and the imagingsystem used will determine the quantity of imaging moiety needed toproduce diagnostic images. In the case of a radioisotope moiety, for ahuman subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibodywill then preferentially accumulate at the location of cells whichcontain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodiesand Their Fragments.” (Chapter 13 in TUMOR IMAGING: THE RADIOCHEMICALDETECTION OF CANCER, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In one embodiment, monitoring of a disease, disorder or infection iscarried out by repeating the method for diagnosing the disease, disorderor infection, for example, one month after initial diagnosis, six monthsafter initial diagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Example 1 Humanization of Mouse Anti-Cd32b Mab 2b6

RNA was converted to cDNA and the VH and VL segments were PCR amplifiedusing the RLM-RACE kit (Ambion, Inc.). Gene specific primers for the VHwere SJ15R, SEQ ID NO:47 (5′ GGT CAC TGT CAC TGG CTC AGG G 3′) andSJ16R, SEQ ID NO:48 (5′ AGG CGG ATC CAG GGG CCA GTG GAT AGA C 3′). Genespecific primers for the VL were SJ17R, SEQ ID NO:49 (5′ GCA CAC GAC TGAGGC ACC TCC AGA TG 3′) and SJ18R, SEQ ID NO:50 (5′ CGG CGG ATC CGA TGGATA CAG TTG GTG CAG CAT C 3′). The RACE product was inserted into theplasmid pCR2.1-TOPO using a TOPO TA Cloning kit (Invitrogen, Inc.). Theresulting plasmids were then subjected to DNA sequencing to determinethe VH and VL sequences for 2B6. The resulting sequences were thentranslated and the predicted amino acid sequence determined for each.From these sequences the framework (FR) and complementarity determining(CDR) regions were identified as defined by Kabat. The mouse VH was thenjoined to a human C-Gamma1 constant region and an Ig leader sequence andinserted into pCI-neo for mammalian expression. The mouse VL was joinedto a human C-kappa segment and an Ig leader sequence and also clonedinto pCI-neo for mammalian expression.

The humanized 2B6 VH consists of the FR segments from the human germlineVH segment VH1-18 and JH6, and the CDR regions of the 2B6 VH. Thehumanized 2B6 VL consists of the FR segments of the human germline VLsegment VK-A26 and JK4, and the CDR regions of 2B6 VL. The humanized VHand VL segments were assembled de novo from oligonucleotides combinedand amplified by PCR. The resulting fragment was then combined by PCRwith a leader sequence and the appropriate constant region segmentcloned into the expression vector pCI-neo as a Nhe I-EcoR I fragment.The DNA sequence of the resulting plasmids was confirmed by sequenceanalysis. For the VL, none of the plasmids analyzed had a perfectlycorrect sequence. The two best inserts were combined to reduce thenumber of incorrect positions, then these positions were corrected bysite-directed mutagenesis. After this procedure light chain segmentshaving predicted humanized 2B6 VL sequence were identified.

The alignment of the amino acid sequences of mouse 2B6 VH, humanized 2B6VH1-18 and human JH6 is shown in FIG. 1A. FIG. 1B shows the alignment ofamino acid sequences of murine 2B6VL, human 2B6VL-1, human 2B6VL-2;human 2B6VL-3, and human Jκ4.

Example 2 Expression and Characterization of the Humanized 2B6 Heavy andLight Chains

The hu2B6 heavy chain (HC) expression plasmid was co-transfectedtogether with ch2B6 light chain (LC) into HEK-293 cells. At the sametime, the ch2B6HC was co-transfected with the ch2B6LC. After three daysin culture the amount of human IgG expressed was quantitated by ELISA.Binding to dimeric soluble FcγRIIb-Fc was then determined by ELISAassay.

Protocol for ELISA assay: 2.5 ng/well of soluble FcγRIIb-Fc was capturedon 96-well Maxisorp plates by mouse anti-FcγRIIb antibody 3H7 at roomtemperature for 1 hour. A serial of two-fold dilution of conditionedmedium of ch2B6 or hu2B6Hc/Ch2B6Lc starting from 25 ng/well was added tothe each well. The plate was incubated at room temperature for 1 hour,then binding was detected by HRP conjugated F(ab′)₂ goat anti human IgGF(ab)′₂ specific secondary antibody. After incubation with the secondaryantibody for approximately 45 minutes, the plate was developed using aTMB substrate. After 5 minutes incubation, the reaction was stopped by1% H₂SO₄. The OD₄₅₀ nm was read by SOFTmax program. Between each step,the plates were washed 3 times with PBS/0.1% Tween20. Plates wereblocked by 0.5% BSA in PBS/0.1% Tween 20 for 30 mins at room temperaturebefore adding soluble FcγRIIb-Fc.

Results: The results of the ELISA assays are depicted in FIG. 2, whichindicate that the hu2B6HC/ch2B6LC mAb bound to the receptor with similaraffinity as the ch2B6HC/ch2B6LC mAb.

FACS analysis was then performed to measure the binding of the mAbs toDaudi cells.

Protocol for FACS analysis. Approximately 10⁶ Daudi cells were used foreach antibody staining. Cells were washed once with PBS. Primaryantibodies (Ch2B6, Hu2B6Hc/ch2B6Lc, human IgG1) were diluted into 0.5,0.1, 0.02 μg/mL in PBS/1% BSA and 100 μL of diluted antibodies wastransferred to the cells. After 30 mins incubation at 4° C., cells werewashed once with 1 mL PBS/1% BSA. PE conjugated F(ab′)₂ fragment of goatanti human IgG Fc specific (Jackson ImmunoReseach, Inc.) was used assecondary antibody at 1:1000 dilution. After 30 mins incubation at 4°C., cells were washed once with 1 mL PBS/1% BSA. The cells were thenresuspended in 500 μL of PBS/1% BSA and subjected to be FACS analysis.

Results: The results indicate that hu2B6HC/ch2B6LC mAb binds to thishuman B cell tumor line with the same affinity as the chimeric mAb(Table 6).

TABLE 6 Primary Antibody Concentration (μg/ml) Mean Fluorescence HumanIgG1 0.5 9.49 0.1 N/A 0.02 N/A Ch2B6 0.5 647.48 0.1 511.85 0.02 172.68Hu2B6Hc/Ch2B6Lc 0.5 648.99 0.1 546.46 0.02 196.93

Transfections of HEK-293 cells were performed using the followingcombinations: hu2B6HC/hu2B6LC, hu2B6HC/ch2B6LC, ch2B6HC/hu2B6LC andch2B6HC/ch2B6LC. After three days in culture the amount of human IgGexpressed was quantitated by ELISA using the protocol described above.Binding to dimeric soluble FcγRIIb-Fc was then determined by ELISA. Theresults of this experiment, depicted in FIG. 3, indicated that all ofthe mAbs bound to the receptor with similar affinity. FACS analysis wasthen performed using the protocol described above to measure the bindingof the mAbs to Daudi cells (Table 7). The results indicate thathu2B6HC/hu2B6LC mAb binds to this human B cell tumor line with the sameaffinity as the ch2B6 mAb.

TABLE 7 Primary Antibody Concentration (ug/ml) Mean Fluorescence HumanIgG1 0.5 6.07 0.1 N/A 0.02 N/A Ch2B6 0.5 551.52 0.1 514.69 0.02 168.17Hu2B6 0.5 628.82 0.1 618.13 0.02 228.74

Example 3 Generation, Expression and Binding of HU2B6LC Variants

There is a consensus sequence of N-glycosylation site (Asn-Xaa-Ser/Thr)in the Hu2B6LC CDR2 region (Asn₅₀-Val-Ser). To eliminate theglycosylation at residue 50 and thus limit potential variation inproduction as well as potential immunogenicity in a pharmaceuticalapplication, other amino acids were substituted at the position 50 usingsite-directed mutagenesis (Stratagene kit). Two different versions ofHu2B6LC were generated, Hu2B6LC-N50Y Hu2B6LC-N50Y,V51A. These aminoacids were chosen because Tyrosine is the human acceptor residue atCDRL2 position 50 and Alanine is the residue at CDRL2 position 51 in thehuman germline gene segment.

Transfections of HEK-293 cells were performed using the followingcombinations: hu2B6HC/hu2B6LC; hu2B6HC/hu2B6LC(N50Y);hu2B6HC/hu2B6LC(N50Y,V51A); ch2B6HC/ch2B6LC. After three days in culturethe amount of human IgG expressed was quantitated by an ELISA assay,using the method described above. Binding to dimeric soluble FcγRIIb-Fcwas determined by ELISA assay. The results of this experiment, depictedin FIG. 4, indicated that all of the mAbs bound to the receptor withsimilar affinity. FACS analysis was then performed to measure thebinding of the mAbs to Daudi cells (Table 8). The results demonstratethat the two variants of hu2B6LC/hu2B6HC mAbs bind to this human B celltumor line with the same affinity as the ch2B6 mAb.

TABLE 8 Primary Antibody Concentration (μg/ml) Mean Fluorescence HumanIgG1 0.5 1.23 0.1 N/A 0.02 N/A Ch2B6 0.5 192.88 0.1 141.01 0.02 45.59Hu2B6 0.5 201.69 0.1 174.37 0.02 58.65 Hu2B6 N50Y 0.5 191.16 0.1 134.560.02 40.14 Hu2B6N50Y, V51A 0.5 167.16 0.1 133.83 0.02 45.95

Example 4 Binding of mAbs to FcRIIA

Protocol for ELISA assay: 100 ng/well of soluble FcγIIA in carbonatebuffer was coated on 96-well Maxisorp plates at 4° C. overnight. Aserial of two-fold dilution of conditioned medium of Ch2B6;hu2B6HC/hu2B6LC; hu2B6HC/hu2B6LC(N50Y); hu2B6HC/hu2B6LC(N50Y,V51A); andpurified IV.3 starting from 25 ng/well was added to the each well. Theplate was incubated at room temperature for 1 hour. The binding wasdetected by HRP conjugated F(ab′)2 goat anti human IgG F(ab′)2 specificsecondary antibody for Ch2B6 and all hu2B6 samples and HRP conjugatedF(ab′)2 goat anti mouse IgG (H+L) secondary antibody for IV.3. Afterincubation with the secondary antibody for approximately 45 minutes, theplate was developed using a TMB substrate. After 5 mins incubation, thereaction was stopped by 1% H₂SO₄. The OD₄₅₀ nm was ready by SOFTmaxprogram. Between each step, the plates were washed 3 times with PBS/0.1%Tween 20. The plates were blocked by 0.5% BSA in PBS/0.1% Tween 20 for30 mins at room temperature before adding the serial diluted antibodies.

Results: These data show that the humanized 2B6 antibody did not loseits ability to selectively bind CD32B during the humanization process(FIG. 5). In summary IV.3 (a murine Mab against FcγIIA) binds FcγIIAwhile chimeric and humanized 2B6 does not.

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and functionally equivalent methodsand components are within the scope of the invention. Indeed, variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

Various references are cited herein, the disclosure of which areincorporated by reference in their entirety.

1. A humanized antibody having an amino acid sequence that comprisesCDRs from a monoclonal antibody which specifically binds theextracellular domain of native human FcγRIIB with a greater affinitythan said antibody binds the extracellular domain of native humanFcγRIIA.
 2. The humanized antibody of claim 1, wherein the antibodycomprises CDRs from monoclonal antibody 2B6 or 3H7.
 3. The humanizedantibody of claim 1, wherein the antibody comprises CDRs from monoclonalantibody 1D5, 2E1, 2H9, 2D11, or 1F2.
 4. The humanized antibody of claim1, wherein the antibody comprises a humanized variable region having aheavy chain region and a light region, said heavy chain region having anamino acid sequence of SEQ ID NO. 24 or SEQ ID NO. 37 and said lightchain region having the amino acid sequence of SEQ ID NO. 18, SEQ ID NO.20, SEQ ID NO. 22 or SEQ ID NO.
 46. 5. The humanized antibody of claim1, wherein the antibody comprises a VH CDR1 having an amino acid of SEQID NO. 1 or SEQ ID NO. 29, a VH CDR2 having an amino acid of SEQ ID NO.2 or SEQ ID NO. 30, and a VH CDR3 having an amino acid of SEQ ID NO. 3or SEQ ID NO.
 31. 6. The humanized antibody of claim 1, wherein theantibody comprises a VL CDR1 having an amino acid of SEQ ID NO. 8 or SEQID NO. 38, a VL CDR2 having an amino acid of SEQ ID NO. 9, SEQ ID NO.10, SEQ ID NO. 11 or 39, and a VL CDR3 having an amino acid of SEQ IDNO. 12 or SEQ ID NO.
 40. 7. The humanized antibody of claim 1 comprisinga variant CDR region, wherein the variant CDR region comprises at leastone amino acid modification in VL CDR2.
 8. The humanized antibody ofclaim 7, wherein the at least one amino acid modification comprises asubstitution at position 50 with tyrosine.
 9. The humanized antibody ofclaim 7, wherein the at least one amino acid modification comprises asubstitution at position 51 with alanine.
 10. The humanized antibody ofclaim 1, wherein the antibody contains no amino acid modification in theframework region.
 11. The antibody fragment of claim 1, wherein saidfragment is a F(ab′)2 fragment or a F(ab) fragment.
 12. The antibody ofclaim 1, wherein said antibody is a single chain antibody.
 13. Theantibody of claim 1, wherein said antibody is operably linked to aheterologous polypeptide.
 14. The antibody of claim 1, wherein saidantibody is conjugated to a therapeutic agent.
 15. The antibody of claim14, wherein said therapeutic agent is a cytotoxin.
 16. The antibody ofclaim 1 which blocks binding of an Ig-Fc to FcγRIIB.
 17. The antibody ofclaim 1, wherein said antibody reduces tumor growth more effectivelythan Rituxin.
 18. An isolated nucleic acid comprising a nucleotidesequence encoding a heavy chain or a light chain of the antibody orfragment thereof of claim
 1. 19-23. (canceled)
 24. A method of treatingcancer in a patient having a cancer characterized by a cancer antigen,said method comprising administering to said patient a therapeuticallyeffective amount of the humanized antibody of claim 1, and an antibodythat specifically binds said cancer antigen and is cytotoxic.
 25. Themethod of claim 24, wherein said cancer is breast, ovarian, prostate,cervical or pancreatic cancer.
 26. The method of claim 24, wherein saidcytotoxic antibody is trastuzumab, rituximab, IC14, edrecolomab,cetuximab, VITAXIN™, Campath 1H/LDP-03, epratuzumab, or ZEVLIN™.
 27. Themethod of claim 24, wherein said cancer antigen is MAGE-1, MAGE-3, BAGE,GAGE-1, GAGE-2, N-acetylglucosaminyltransferase, p15, beta-catenin,MUM-1, CDK4, HER-2/neu, human papillomavirus-E6, humanpapillomavirus-E7, or MUC-1.
 28. The method of claim 24, wherein saidcancer antigen is a breast, ovarian, prostate, cervical, or pancreaticcarcinoma antigen.
 29. The method of claim 24 further comprising theadministration of one or more additional cancer therapies.
 30. Themethod of claim 29, wherein said additional cancer therapy is selectedfrom the group consisting of chemotherapy, immunotherapy, radiationtherapy, hormonal therapy, or surgery.
 31. The method of claim 24,wherein said patient is human.
 32. A pharmaceutical compositioncomprising: (i) a therapeutically effective amount of the humanizedantibody of claim 1; (ii) a cytotoxic antibody that specifically binds acancer antigen; and (iii) a pharmaceutically acceptable carrier. 33-42.(canceled)
 43. A method of enhancing an antibody mediated cytotoxiceffect in a subject being treated with a cytotoxic antibody, said methodcomprising administering to said patient the humanized antibody of claim1 in an amount sufficient to enhance the cytotoxic effect of saidcytotoxic antibody. 44-46. (canceled)
 47. A method of treating cancer ina patient having a cancer characterized by a cancer antigen, said methodcomprising administering to said patient a therapeutically effectiveamount of the humanized antibody of claim 1, wherein the administeredantibody reduces the population of cancer cells expressing FcγRIIB. 48.A method of treating cancer in a patient having a cancer characterizedby a cancer antigen, said method comprising administering to saidpatient a therapeutically effective amount of the humanized antibody ofclaim 1, wherein the administered antibody eliminates cancer cellsexpressing FcγRIIB.